Using Monoclonal Antibody to Determine Lead Ions

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Vaccine(2008)26,460—468Intranasal administration of a recombinant adenovirus expressing the norovirus capsidprotein stimulates speciﬁc humoral,mucosal,and cellular immune responses in miceLi Guo a,1,Jianwei Wang a,b,∗,1,Hongli Zhou a,b,Hongli Si a,c,Min Wang a, Jingdong Song b,Bingjuan Han b,Yi Shu a,Lili Ren b,Jianguo Qu b,Tao Hung a,ba National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing100052,Chinab State Key Laboratory of Molecular Virology and Genetic Engineering,Institute of Pathogen Biology,Chinese Academy of Medical Sciences,9#Dong Dan San Tiao,Beijing100730,Chinac North-western A&F University,Yangling712100,ChinaReceived18May2007;received in revised form19October2007;accepted18November2007Available online4December2007KEYWORDSNorovirus capsidprotein;Adenovirus vector;Immune responseSummary Norovirus(NV)is a major cause of acute,epidemic nonbacterial gastroenteritis inindividuals of all ages.The immunological mechanism of NV infection and the approaches usedto prevent infection remain to be elucidated.In this study,the speciﬁc immune responses ofBALB/c mice were assessed following intranasal immunization with a recombinant adenovirusvector expressing the genogroup II4(GGII/4)norovirus capsid protein.Analysis of IgM,IgG,andIgA antibodies speciﬁc for the recombinant virus-like particles(VLPs)of NV demonstrated that ahigh level of humoral immunity developed following immunization.Mucosal immune responseswere also detectable in stool,intestinal homogenates,lung homogenates,and lung lavage sam-ples.Speciﬁc cellular immune responses were observed in NV VLPs-restimulated splenocytesby ELISPOT and Th1/Th2cytokine cytometric array(CBA).Serum IgG subclass analysis showedthat a balanced Th1-and Th2-like cellular immune response was induced in BALB/c mice follow-ing immunization with recombinant adenovirus.Theseﬁndings demonstrate that the intranasalimmunization of a recombinant adenovirus expressing the NV capsid protein is an efﬁcient strat-egy to stimulate systemic,mucosal,and cellular Th1/Th2immune responses in mice,and couldserve as a novel approach for designing NV vaccines.©2007Elsevier Ltd.All rights reserved.∗Corresponding author at:State Key Laboratory of Molecular Virology and Genetic Engineering,Institute of Pathogen Biology,Chinese Academy of Medical Sciences,9#Dong Dan San Tiao,Beijing100730,China.T el.:+861065105188;fax:+861065105188.E-mail address:wangjw28@(J.Wang).1These authors contributed equally to this work.0264-410X/$—see front matter©2007Elsevier Ltd.All rights reserved.doi:10.1016/j.vaccine.2007.11.039Immunity against norovirus induced by recombinant adenovirus461IntroductionNorovirus(NV),or Norwalk-like virus,is a genus of the Caliciviridae family and is recognized as a major cause of acute,epidemic nonbacterial gastroenteritis in individuals of all ages[1,2].NV-related gastroenteritis outbreaks usu-ally occur in families,schools,nursing homes,hospitals,and military facilities through contact with contaminated food and water[2—4].While NV gastroenteritis is a mild self-limiting illness,it is associated with a high incidence of morbidity and hospitalization even in industrialized coun-tries.Recent studies have illustrated that∼90%of acute nonbacterial gastroenteritis outbreaks in the United States were caused by NV[5—7].In England and Wales,NV is responsible for almost50%of gastroenteritis outbreaks[8]. Finland,Sweden,Netherlands,Germany,and Japan have similar infectivity rates[9—12].In developing countries,NV-associated disease also takes an economic toll,especially in the young and very old[13,14].For these reasons,it is important to develop an effective NV vaccine especially for these at-risk populations[15].NV is composed of a7.4—8.3kb positive-sense,single-stranded RNA genome with three open reading frames (ORFs)[16,17].ORF1encodes a non-structural polyprotein that contains the3D RNA-dependent RNA polymerase region, ORF2encodes the viral capsid(VP-1),and ORF3encodes a minor basic virion protein(VP-2)[18].ORF2expres-sion by recombinant baculovirus in insect cells[19]or by the Venezuelan equine encephalitis virus replicon vector in mammalian cells[20,21]yields self-assembled virus-like particles(VLPs).These recombinant NV VLPs are morpho-logically and antigenically similar to the wild-type virus [20,21].Since cell culture systems and animal models of NVs are lacking[2],little is known about the details of the anti-NV immune response and few approaches have been designed to stimulate long-term,stable Th1/Th2-balanced immunity that would be required for a reliable prophylaxis. Recombinant VLPs may thus serve as an important source of antigen to study anti-NV immunity.Recent studies sug-gest that rNV VLPs can elicit systemic and mucosal immune responses in CD1and BALB/c mice,indicating that they may serve as a potential mucosal vaccine against NV[21—24]. However,these responses were only achieved by the addi-tion of mucosal adjuvants such as cholera toxin(CT)or the mutant E.coli heat-labile toxin,LT(R192G)[21—23].Since such adjuvants cannot be used in humans as a result of their potential toxicity,it is important to develop other immu-nization approaches to vaccine development.In this report,we determined whether intranasal immu-nization of mice with a recombinant adenovirus,rvAdGGII4, expressing the NV capsid,could elicit a strong anti-NV immune response,and illustrated a potential new approach for NV vaccine development.Materials and methodsPreparation of the recombinant adenovirus expressing NV capsid proteinThe GGII/4NV capsid protein gene from Lordsdale virus (Genbank X86557)was codon-optimized and artiﬁcially syn-thesized by adopting codons that were used in insect cells as described previously[25].The recombinant adenovirus, rvAdGGII4,was generated in HEK293cells using the AdEasy adenoviral vector system(Stratagene,Cedar Creek,TX) according to the manufacturer’s protocol.NV capsid pro-tein expression was conﬁrmed by Western blot using an anti-NV capsid monoclonal antibody(Guo L.et al.,unpub-lished data).The recombinant adenovirus was puriﬁed by cesium chloride density gradient centrifugation[26],titered with an Adeno-X TM Rapid Titer Kit(BD Biosciences Clon-tech,Mountain View,CA)and stored at−70◦C prior to use.Preparation of NV-like particlesNV VLPs were prepared and puriﬁed as previously described, with minor modiﬁcations[27].The optimized GGII/4NV cap-sid gene was cloned into the baculovirus transfer vector, pFastBac1(Invitrogen,Carlsbad,CA)and a recombinant baculovirus was generated in Sf9cells using the Bac-to-Bac®Baculovirus Expression System(Invitrogen,Carlsbad,CA) protocol provided by the manufacturer.Recombinant bac-ulovirus infected Sf9cell supernatants were collected5days post-infection and cellular debris was removed by centrifu-gation(20min at10,000rpm).VLPs were precipitated from the clariﬁed supernatants using PEG6000(6%),and the pel-lets were sonicated and centrifuged at35,000rpm through a40%sucrose cushion for3h.Puriﬁed VLPs were conﬁrmed by Western blot using an anti-NV capsid monoclonal antibody [25].Protein concentrations were determined using the BCA Protein Assay Reagent Kit(Pierce,Rockford,IL)and stored at−70◦C prior to use.Animal immunization and sample collectionSix-to8-week-old female BALB/c mice,purchased from the Institute of Laboratory Animal Sciences at the Chinese Academy of Medical Sciences,were used for all immuniza-tions and maintained in Animal Biosafety Level-2facilities. After intranasal anesthetization with ether,mice were intranasally(i.n.)inoculated with1×106ifu(infectious units)(in0.1ml)of the recombinant adenovirus,rvAdG-GII4(rvAdGGII4group),1×106ifu(in0.1ml)of the empty recombinant adenovirus serotype5vector,which contained no insert(rvAd5-empty group,negative control),or0.1ml phosphate-buffered saline(PBS;PBS group,blank control) at day0.All mice were boosted at days14and28.Serum and fecal samples from all groups were col-lected at days0,14,28,and35post-inoculation(dpi),and stored at−20◦C prior to testing.At35dpi,the mice were sacriﬁced,and lung lavages,lung homogenates,and intesti-nal homogenates were used to measure mucosal immune responses as previously described[28]with minor modiﬁca-tions.In brief,the lungs and20-cm sections of the small intestines excluding the Peyer’s patches were ground into homogenates using a high-speed tissue grinder,suspended in PBS(pH7.2)to produce a10%(w/v)suspension,and cen-trifuged at3000rpm for5min.The supernatants and lung lavages were stored at−70◦C prior to use.Splenocytes were also isolated and restimulated in order to measure cellular immune responses.462L.Guo et al.Analysis of NV-speciﬁc antibody production Antibodies in the sera,fecal matter,intestinal homogenates, lung homogenates,and lung lavages were measured by indi-rect ELISA.Microtiter plates(Costar,Bethesda,MD)were coated with0.1␮g VLPs from GGII/4NV per well at4◦C overnight.The plates were blocked with1%(w/v)BSA in PBS at37◦C for2h.The fecal samples were diluted into a10%(w/v)suspension in PBS.Serum and fecal extracts were twofold serially diluted using0.1%BSA in PBS.Intesti-nal homogenates,lung homogenates,and lung lavages were diluted1:10.Samples were added to the wells and incubated for1h at37◦C.After washingﬁve times with0.05%T ween 20in PBS(PBS-T),the plates were incubated at37◦C for1h using a1:5000dilution of horseradish peroxidase-conjugated goat anti-mouse IgG,IgA,IgM,IgG1,or IgG2a(Sigma, St.Louis,MO).Plates were washed and developed with 100␮l/well of0.1mg/ml3,3 ,5,5 -tetramethylbenzidine (TMB;Sigma,St.Louis,MO)peroxidase substrate containing 0.03%hydrogen peroxide.Color development was stopped by the addition of50␮l/well of2M sulfuric acid.The absorbency at450nm(A450)was determined using an ELISA plate reader(BioRad550,Hercules,CA).IFN-␥ELISPOT assayMurine spleens were removed and ground under sterile conditions using a5-ml syringe plunger,and splenocytes were isolated with Mouse Lymphoprep(Dakewe,Shenzhen, China).The freshly made splenocytes were washed twice and resuspended in R10medium[RPMI1640supplemented with10%fetal bovine serum(FBS),100IU/ml penicillin, and100␮g/ml streptomycin(Hyclone,Logan,UT)].IFN-␥ELISPOT assays were performed using an ELISPOT Set(BD Biosciences,San Diego,CA)as recommended by the manu-facturer.In brief,5×105freshly isolated splenocytes were added to each of two replicate wells coated with anti-mouse IFN-␥monoclonal antibody(MAb)(BD Biosciences,Catalog No.51-2525kc)and stimulated with20␮g/ml VLPs at37◦C in a5%CO2incubator for24h.Unstimulated splenocytes were used to measure background cytokine production.The cells were then lysed with deionized(DI)water,and the plates were incubated at room temperature with biotinylated IFN-␥antibody(BD Biosciences,Catalog No.51-1818kz)for2h, and peroxidase-labeled streptavidin(BD Biosciences,Cata-log No.51-9000209)for1h.After washing with PBS,100␮l of theﬁnal substrate solution(BD Biosciences,Catalog No. 551951)was added to each well and spot development was monitored.The plates were washed with distilled water to stop the reaction.IFN-␥spot-forming cells(SFC)were counted using an automated ELISPOT plate reader system. Results were expressed as the number of SFC/106cells. Assay for multi-cytokine productionFreshly isolated murine splenocytes were cultured in a96-well,round-bottom tissue culture plate at5×105cells/well in R10medium and stimulated with NV VLPs for48h.Super-natants were collected and IL-2,IL-4,IL-5,TNF-␣,and IFN-␥levels were quantitated using the mouse Th1/Th2Cytokine Cytometric Array Bead(CBA)Kit(BD PharMingen,San Diego,CA)according to the manufacturer’s protocol.The data were acquired with a FACSCalibur®ﬂow cytometer(BD Bio-sciences,San Jose,CA)using2-color detection and analyzed using CBA software(BD PharMingen).Analysis of anti-adenoviral serotype5(Ad5) immunityThe anti-Ad5serum IgG,IgM,and IgA antibodies were ana-lyzed by indirect ELISA as described in Section of Analysis of NV-speciﬁc antibody production.The microtiter plates (Costar,Bethesda,MD)were coated with1×105ifu(in 0.1ml)of wild serotype5adenovirus,which was puri-ﬁed using the CsCl2cushion ultra-centrifuge[26],at4◦C overnight.Statistical analysisSerum and fecal antibody titers were log10transformed, expressed as geometric mean titers(GMT s),and com-pared using the Student’s t-test.The Wilcoxon rank sum test was used to compare intestinal homogenates,lung homogenates,and lung lavage antibody levels between groups.ELISPOT and CBA results between groups were compared using the Student’s t-test.Nonresponders were included in all calculations.The lowest serum and fecal sample dilution(1:50)was divided by two and used as the negative sample titer(i.e.,negative samples were assigned a titer of25).All tests were two-tailed,and a value of p<0.05was considered statistically signiﬁcant.ResultsHumoral immune responses in miceT o determine whether the recombinant adenovirus,rvAdG-GII4,could stimulate speciﬁc immune responses following i.n.administration,BALB/c mice were immunized three times on days0,14,and28,respectively.The control groups were treated with rvAd5-empty or PBS accord-ing to the same scheme.All preimmune serum titers of IgM,IgA,and IgG antibodies against norovirus VLPs or adenovirus were negative(<50)(data not shown).In addi-tion,each of the control mice remained negative for VLP antibodies at35dpi.After theﬁrst i.n.administra-tion of rvAdGGII4,however,100%of the mice had strong speciﬁc serum IgM titers(GMT=315.9).The GMT of IgM peaked(GMT=348.2)after the second immunization(28dpi) and decreased(GMT=219.1)after the third immunization (35dpi)(Fig.1A).Serum IgG responses were mounted by allﬁve mice at day14post-immunization with rvAdGGII4 (GMT=3816,ranging from3200to10,000).After the sec-ond(28dpi)and the third immunizations(35dpi),IgG titers continued to increase,with GMT s of155,261and632,978, respectively(Fig.1B).No mice had detectable levels of serum IgA antibodies by14dpi.Serum-speciﬁc IgA anti-body titers were detected in all groups after the second immunization(GMT=377.6,ranging from300to800)and increased after the third immunization(GMT=2009.5,rang-ing from1600to3200)(Fig.1C).IgM,IgG,and IgA responsesImmunity against norovirus induced by recombinant adenovirus463Figure 1Humoral responses in mice sera.BALB/c mice were immunized as described in Section 2.Blood samples were col-lected at 14,28,and 35dpi,respectively .Serum-speciﬁc IgM (A),IgG (B),and IgA (C)antibodies from individual mice were examined by ELISA and used to calculate the GMT for each group.For the IgM-negative,IgG-negative,and IgA-negative (<50)control samples,titers of 25were used to calculate the GMT .Days post-immunization schedule are shown on the X-axis.Error bars represent the standard errors of the means.Above each bar is the number of responders over the total number of mice tested.were undetectable in mice treated with the rvAd5-empty vector or PBS.These data indicate that rvAdGGII4stimu-lates a strong NV speciﬁc humoral response and that serum antibodies were substantially enhanced following rvAdGGII4immunization.Figure 2Fecal IgA and IgG responses in BALB/c mice.Murine fecal samples were collected at 35dpi.Levels of speciﬁc IgG and IgA antibodies in the feces were measured by ELISA and used to calculate the GMTs.The error bars show the standard errors of the mean.The number above each bar depicts the number of responders over the total number of animals tested.Serum IgG subclass analysisAnti-VLPs IgG1and IgG2a antibodies were measured in NV VLP-speciﬁc IgG positive sera (T able 1).The increase in IgG1and IgG2a levels coincided with the rise in serum IgG.Sig-niﬁcantly high titers of both IgG1and IgG2a were observed following administration of rvAdGGII4,and there was no signiﬁcant difference between these class types (p >0.05,Student’s t -test).Thus,the Th1-(IgG2a)and Th2-like (IgG1)immune responses were roughly balanced in rvAdGGII4-immunized mice.Mucosal responses in miceT o evaluate whether recombinant adenovirus could stimu-late speciﬁc mucosal responses,fecal suspensions,intestinal homogenates,lung homogenates,and lung lavages were prepared from immunized mice.Mucosal immune responses to NV VLPs were observed in all vaccinated mice fol-lowing primary i.n.administration of 1×106ifu rvAdGGII4and after each rvAdGGII4boost immunization.All immu-nized mice (5/5)had NV VLP-speciﬁc fecal IgG and IgA responses (GMT =470.43and 819.07,respectively),while the rvAd5-empty and PBS groups remained nega-tive (titers <50)(Fig.2).IgA and IgG levels in intestinal homogenates,lung homogenates,and lung lavages were evaluated at a single dilution (1:10),and results were expressed as A 450values.Strong IgA and IgG responses were induced in the intestinal homogenates (p <0.05for IgA,p <0.001for IgG,respectively),lung homogenates (p <0.001for IgA and IgG,respectively)and lung lavage samples (p <0.001for IgA and IgG,respectively)(Fig.3).TheseTable 1Analysis of serum Ig antibody isotypes to norovirus VLPsImmunogenDays post-immunizationGeometric mean titer IgG1/IgG2aIgG1IgG2a rvAdGGII435201189.380546.30.539rvAd5-empty 35<50<50PBS 35<50<50464L.Guo etal.Figure3Levels of speciﬁc IgA(A)and IgG(B)antibodies in the intestines and lungs of BALB/c mice immunized with rvAdGGII4. At days14and28following the primary immunization,each group of mice was administered the same amount of antigen, and intestinal homogenates,lung homogenates,and lung lavage were obtained from each animal1week later.Levels of speciﬁc IgA and IgG antibodies were examined by ELISA.The results are represented as the mean OD(450nm)that was detected in individual mice at a1:10dilution.Error bars represent the standard errors of the mean.data suggest that rvAdGGII4elicits strong mucosal immune responses.IFN-␥ELISPOT responses and cytokine assaysT o measure the cellular immune responses elicited by i.n. administration of rvAdGGII4,the speciﬁc T-cell responses against NV VLPs were monitored in immunized mice by IFN-␥ELISPOT.The mean number of SFC/106in the rvAdGGII4 group was108±31.No spots were detected in unstimulated splenocytes from the rvAdGGII4group.The level of SFC/106 in the rvAd5-empty and PBS control groups were14±2and 9±7,respectively.The differences between the rvAdGGII4 group and the rvAd5-empty or PBS groups was statistically signiﬁcant(p<0.01and0.001,respectively)(Fig.4).These ﬁndings suggest that mice receiving the rvAdGGII4vector developed cellular immune responses to the NV capsid pro-tein.T o further characterize the cellular immune response, rvAdGGII4immunized splenocytes were isolated and T-cell responses were assessed using a Mouse Th1/Th2Cytokine Cytometric Bead Array system.This allows simultaneous measurement of TNF-␣,IFN-␥,IL-5,IL-4,and IL-2lev-els in antigen-stimulated T-cell supernatants.After the mice were immunized with rvAdGGII4and stimulated with NV VLPs,IFN-␥,TNF-␣,and IL-5production were notably high(217.71pg/ml,156.5pg/ml and265.27pg/ml,respec-tively),while IL-2and IL4levels were only moderately increased(32.2pg/ml and27.59pg/ml,respectively).TNF-Figure4IFN-␥ELISPOT assays of recombinant norovirus VLP-stimulated splenocytes following rvAdGGII4immunization. Splenocytes were isolated7days after theﬁnal immunization and restimulated with20␮g/ml VLP for24h.IFN-␥-producing cells were quantitated using an ELISPOT reader.Results were expressed as the average number of SFC per million input splenocytes.Error bars represent the standard errors of the mean.␣,IFN-␥,IL-5,IL-4,and IL-2levels were signiﬁcantly higher in splenocyte supernatants from rvAdGGII4immu-nized mice than from rvAd5-empty vector(33.63pg/ml, p≤0.001;2.43pg/ml,p<0.001;0pg/ml,p<0.001;0pg/ml, p<0.001and 3.09pg/ml,p<0.05,respectively)or PBS-treated mice(36.22pg/ml,p<0.05;1.22pg/ml,p<0.001;0.42pg/ml,p<0.001;0pg/ml,p<0.001and 1.19pg/ml, p<0.01,respectively)(Fig.5).Since IFN-␥,TNF-␣,and IL-2 are markers of a Th1response,while IL-5and IL-4are mark-ers of a Th2response,these results indicated that the Th1-and Th2-like cellular immune responses were both stimu-lated by i.n.administration of rvAdGGII4.Anti-Ad5antibodies in miceT o determine whether the recombinant adenovirus,rvAdG-GII4,could elicit anti-Ad5immune responses following i.n. administration,anti-Ad5serum antibody levels were mea-sured by indirect ELISA.All immunized mice hadanti-Ad5 Figure5Cytokines detected in splenocyte cultures from mice immunized with rvAdGGII4.Splenocytes were isolated7days after theﬁnal immunization and stimulated with NV VLPs for 48h.TNF-␣,IFN-␥,IL-5,IL-4,and IL-2levels were measured in the culture supernatant using the Cytometric Bead Array. Signiﬁcant differences were observed between the rvAdGGII4 and rvAd5-empty or PBS group.Error bars indicate the standard errors of the mean.Immunity against norovirus induced by recombinant adenovirus465Figure6Anti-Ad5immunity in mice after rvAdGGII4immu-nization.Blood samples were collected at14,28,and35dpi, respectively.Anti-Ad5antibodies were assessed by ELISA and used to calculate the GMT s.The days post-immunization are shown on the X-axis,and the error bars reveal the standard errors of the mean.The number above each bar depicts the number of responders over the total number of animals tested.serum IgG responses following the primary i.n.rvAdGGII4 administration and boost(GMT=299.3,1912.7and6964.4, respectively)(Fig.6).The IgM responses were not observed in mice after theﬁrst administration.The GMT of IgM was 65.9after the second immunization(28dpi),and decreased to<50after the third immunization(35dpi)(Fig.6).While no detectable anti-Ad5IgA antibodies were found in the sera after theﬁrst and second immunizations,anti-Ad5serum-speciﬁc IgA antibody titers could still be detected in mice after the third immunization(GMT=269.9)(Fig.6).DiscussionSince NV infections are localized in the gastrointestinal tract,local mucosal immunity is believed to play an impor-tant role in protection against infection.It is accepted that an ideal NV vaccine would induce strong mucosal immu-nity[29].In previous studies,anti-NV immunity using VLPs primarily relied on the adjuvants like enterotoxin-based LT or CT,since VLPs are too weak to stimulate immune responses,especially within the mucosa.However,CT and LT are potentially toxic to the human central nervous sys-tem[30].An intranasal inﬂuenza vaccine containing LT as a mucosal adjuvant,which resulted in46cases of Bell’s palsy in Switzerland,was recently removed from the mar-ket[31],indicating that CT or LT adjuvants are not suitable for humans.Thus,it is important to develop a novel immu-nization strategy that does not require the use of mucosal adjuvants.The replication-defective adenovirus serotype5vector is recognized as an attractive vector in both gene ther-apy and novel genetic vaccine development[32].Its safety and efﬁcacy is shown in numerous animal experiments and human clinical trials,and two adenovirus-based gene ther-apy drugs are licensed.Adenovirus vectors have been used in studies of potential Ebola virus,human immunodeﬁ-ciency virus type1(HIV-1),inﬂuenzavirus,and tuberculosis vaccines[33—39].Clinical trials using adenoviral-vectored vaccines against inﬂuenza virus,HIV-1,some malignant tumors,and other pathogens are currently in progress [40—43].These clinical trials show that adenoviral vector-based vaccines are safe,effective,and have no known toxicity or inﬂammatory properties.Adenovirus vectors are also shown to boost CTL responses against heterologous gene products[44—47].However,existing anti-adenoviral immune responses impair foreign antigen expression after repeated intramuscular immunizations.Since humans are widely infected by adenoviruses and usually possess high anti-adenovirus antibody levels,existing immunity often limits application in cases where repeated immunization is required.Recently,intranasal immunization has become an attractive method for inducing multiple immunities.Thus, we tested the immunogenicity of the NV capsid protein after repeated i.n.administration.We illustrated that sig-niﬁcant serum,mucosal,and cellular immune responses against NV VLPs were elicited after mice were immu-nized with the recombinant adenovirus.Serum IgM and IgG responses against NV VLPs rose remarkably after the ﬁrst rvAdGGII4inoculation,and IgM,IgG,and IgA antibody titers increased to high levels after the third immunization. IgA and IgG responses were also observed in fecal sam-ples,intestinal homogenates,lung homogenates,and lung samples.Theseﬁndings suggest that the i.n.route is an efﬁ-cient pathway to evoke NV capsid-speciﬁc immunity by an adenovirus vector and the regionalization of the mucosal immune system allows immunizations in the gut by deliver-ing a vaccine through the intranasal route.The results also show that repeated i.n.administration of the adenovirus vector effectively boosted NV capsid-speciﬁc serum antibod-ies,demonstrating that existing anti-adenoviral immunity did not impair the recombinant adenovirus-speciﬁc immune response.Theseﬁndings are similar to our results using an adenovirus vectored rotavirus antigen[48].Unexpectedly,intestinal homogenates from the rvAd5-empty and PBS control-treated mice produced low SIgA levels.We speculate that there may have been low SIgA levels in the mouse intestine.The intestine is the largest immunological organ,housing70—80%of all immunoglobulin-producing cells and producing large amounts of SIgA(50—100mg/kg body weight)[49].High background on the ELISA assay may be attributed to the interference of intestinal SIgA or other intestinal contents.Previous studies showed that cytotoxic T lymphocytes (CTLs)play an important role in preventing viral diseases and clearing viruses like hepatitis C[50].Cellular immune response may also be crucial for disease prevention,as well as control and clearance of NV infection.Lindesmith et al.ﬁrst characterized the Th1and Th2cellular immune responses to a challenge with Snow Mountain virus[51]. Recent studies showed that BALB/c mice that were i.n. immunized with VLPs and the mucosal adjuvants,LT or LT (R192G),induced speciﬁc Th1/Th2-like immune responses [27].However,there has been little information regarding the anti-NV cellular immune response.T o measure the cel-lular immune responses in rvAdGGII4-immunized mice,we measured NV VLP-speciﬁc T-cell responses using the IFN-␥ELISPOT assay.Our results indicate that the recombinant adenovirus,rvAdGGII4,was able to induce a strong cellular immune response.Importantly,both Th1-and Th2-like cel-lular immune responses were induced in rvAdGGII4-treated mice.It is well known that Th1-associated cytokines help to regulate antiviral cellular responses,while Th2-associated cytokines enhance humoral immune responses.In our study, TNF-␣and IFN-␥secretion were signiﬁcantly increased,and IL-2levels were moderately increased.At the same time,466L.Guo et al.the Th2cytokine,IL-5,which promotes IgA production, was signiﬁcantly elevated,aﬁnding consistent with high serum and mucosal IgA antibody titers.IL-4levels were also bined with analysis of serum IgG1/IgG2a lev-els,these data suggest that a roughly balanced Th1/Th2 immune response is induced by the adenovirus vectored NV capsid.Due to the unavailability of an animal model of viral chal-lenge,it remains unknown whether the immunity evoked by i.n.adenovirus immunization is capable of protecting against NV infection.However,we may anticipate that it would have a protective effect based on knowledge obtained from studies of other viral infections like rotavirus,which also infect the intestinal surface.Evidence suggests that high local mucosal immune responses against rotavirus are critical for resisting viral infection.Previous studies showed that serum and fecal IgA antibodies are associated with protection against rotavirus reinfection[52,53].Moreover, about a recent study showed that30—40%of volunteers developed mucosal anti-VLP IgA after oral NV VLP immuniza-tion[54].Thus,it is reasonable to speculate that high levels of immunity stimulated by recombinant adenoviruses play an important role in preventing NV infection[21].However, the precise level of protection will require further challenge experiments using volunteer or surrogate challenge models, such as recombinant vaccinia virus,that have been used to study hepatitis C virus(HCV)[55]and hantaan virus infec-tion[56].Further studies will also be required to determine the duration of immunity,and the relationships between the three immune responses,including their cross-protective role with other noroviruses.Although the regionalization of the mucosal immune sys-tem permits gut immunizations through intranasal delivery of a vaccine,swallowing of the inoculum by the vaccinated animals cannot be excluded.Since NV attacks and repli-cates in the small intestine,local immunization of the gut may help to protect the host from NV infection.The experi-ence of Adenovirus4and7in the US Army showed that oral immunization is safe and can evoke strong immune responses over the past four decades[57].Thus,oral immunization may be a potential way to stimulate NV-speciﬁc immunities using recombinant adenovirus immunization.In addition, oral immunization is easier to manage and may be better accepted by children.A recent study showed that oral immu-nization of NV VLPs can produce cross-reactive monoclonal antibodies[58].Thus,the feasibility of oral immunization using the NV capsid protein-expressing recombinant aden-ovirus should be addressed.AcknowledgementsWe are grateful to Dr.Zhendong Zhao(Peking University)for critical reading of the manuscript,Drs.Li Ruan and Xian-grong Qi(National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Preven-tion)for their assistance in ELISPOT assay,and Ms.Shan Mei and Li Li(Institute of Pathogen Biology,Chinese Academy of Medical Sciences)for their assistance in FACS assays.This research was supported by grants from the Chinese Key T ech-nologies R&D Program(No.2003BA712A03—04)and China Nature Science Foundation(No.30700741).References[1]Hedberg CW,Osterholm MT.Outbreaks of food-borneand waterborne viral gastroenteritis.Clin Microbiol Rev 1993;6(3):199—210.[2]Kapikian AZ,Estes MK,Chanock RM.Norwalk group of viruses.In:Fields B,editor.Fields Virology,vol.2,3rd ed.1996.p.783—810.[3]Arness MK,Feighner BH,Canham ML,T aylor DN,Monroe SS,Cieslak TJ,et al.Norwalk-like viral gastroenteritis outbreak in U.S.Army trainees.Emerg Infect Dis2000;6(2):204—7.[4]Koopmans M,Vinjeˇıde Wit M,Leenen I,van der Poel W,van Duynhoven Y.Molecular epidemiology of human enteric caliciviruses in The Netherlands.J Infect Dis2000;181(Suppl2):S262—9.[5]Fankhauser RL,Noel JS,Monroe SS,Ando T,Glass RI.Molecular epidemiology of‘‘Norwalk-like viruses’’in out-breaks of gastroenteritis in the United States.J Infect Dis 1998;178(6):1571—8.[6]Glass RI,Noel J,Ando T,Fankhauser R,Belliot G,MountsA,et al.The epidemiology of enteric caliciviruses from humans:a reassessment using new diagnostics.J Infect Dis 2000;181(Suppl2):S254—61.[7]Shieh Y,Monroe SS,Fankhauser RL,Langlois GW,Burkhardt IIIW,Baric RS.Detection of norwalk-like virus in shellﬁsh impli-cated in illness.J Infect Dis2000;181(Suppl2):S360—6.[8]Dedman D,Laurichesse H,Caul EO,Wall PG.Surveillance ofsmall round structured virus(SRSV)infection in England and Wales,1990—1995.Epidemiol Infect1998;121:139—49.[9]Lew JF,Valdesuso J,Vesikari T,Kapikian AZ,Jiang X,EstesMK,et al.Detection of Norwalk virus or Norwalk-like virus infections in Finnish infants and young children.J Infect Dis 1994;169:1364—7.[10]Hedlund KO,Rubilar-Abreu E,Svensson L.Epidemiology ofcalicivirus infections in Sweden,1994—1998.J Infect Dis 2000;181(2):S275—80.[11]Schreier E,Doring F,Kunkel U.Molecular epidemiology ofoutbreaks of gastroenteritis associated with small round structured viruses in Germany in1997/98.Arch Virol 2000;145:443—53.[12]Inouye S,Yamashita K,Yamadera S,Yoshikawa M,Kato N,OkabeN.Surveillance of viral gastroenteritis in Japan:pediatric cases and outbreak incidents.J Infect Dis2000;181(2):S270—4. [13]Pujol FH,Vasquez G,Rojas AM,Fuenmayor ME,Loureiro CL,Perez-Schael I,et al.Norwalk virus infection in Venezuela.Ann Trop Med Parasitol1998;92(2):205—11.[14]Parks CG,Moe CL,Rhodes D,Lima A,Barrett L,T seng F,et al.Genomic diversity of‘‘Norwalk like viruses’’(NL Vs): pediatric infections in a Brazilian shantytown.J Med Virol 1999;58(4):426—34.[15]Estes MK,Ball JM,Guerrero RA,Opekun AR,Gilger MA,PachecoSS,et al.Norwalk virus vaccines:challenges and progress.J Infect Dis2000;181(Suppl2):S367—73.[16]Xi JN,Graham DY,Wang KN,Estes MK.Norwalk virus genomecloning and characterization.Science1990;250(4987):1580—3.[17]Jiang X,Wang M,Wang K,Estes MK.Sequence and genomicorganization of Norwalk virus.Virology1993;95(1):51—61. [18]Zintz C,Bok K,Parada E,Barnes-Eley M,Berke T,Staat MA,et al.Prevalence and genetic characterization of caliciviruses among children hospitalized for acute gastroenteritis in the United States.Infect Genet Evol2005;5(3):281—90.[19]Jiang X,Wang M,Graham DY,Estes MK.Expression,self-assembly,and antigenicity of the Norwalk virus capsid protein.J Virol1992;66(11):6527—32.[20]Baric RS,Yount B,Lindesmith L,Harrington PR,Greene SR,T seng FC,et al.Expression and self-assembly of norwalk virus capsid protein from Venezuelan equine encephalitis virus repli-cons.J Virol2002;76(6):3023—30.。

herceptin合成路线

herceptin合成路线## Herceptin Synthesis Route.### Introduction.Herceptin is a monoclonal antibody used to treat HER2-positive breast cancer and other types of cancer. It works by targeting and blocking the HER2 protein, which is overexpressed in many types of cancer.The synthesis of Herceptin is a complex process that involves several steps. The following is a general overview of the synthesis route:1. Cell culture: The first step is to culture the Chinese hamster ovary (CHO) cells that will produce Herceptin. These cells are grown in a bioreactor under controlled conditions.2. Transfection: The CHO cells are then transfectedwith a plasmid containing the DNA sequence for Herceptin. This plasmid directs the cells to produce Herceptin.3. Expression: The transfected cells are then allowed to express Herceptin for several days.4. Purification: The Herceptin is then purified from the cell culture supernatant using a series of chromatography and filtration steps.5. Formulation: The purified Herceptin is then formulated into a solution that is suitable for injection.## Process Details.Cell Culture.CHO cells are used to produce Herceptin because they are able to produce high levels of recombinant proteins. The cells are grown in a bioreactor under controlled conditions of temperature, pH, and dissolved oxygen.Transfection.The CHO cells are transfected with a plasmid containing the DNA sequence for Herceptin. This plasmid is typically introduced into the cells using a lipofection reagent.Expression.The transfected cells are then allowed to express Herceptin for several days. During this time, the cells produce high levels of Herceptin, which is secreted into the cell culture supernatant.Purification.The Herceptin is then purified from the cell culture supernatant using a series of chromatography and filtration steps. These steps remove impurities and concentrate the Herceptin.Formulation.The purified Herceptin is then formulated into a solution that is suitable for injection. This solution typically contains a buffer, a stabilizer, and a preservative.中文回答：## 赫赛汀合成路线。

MSDS(单克隆抗体)

MATERIAL SAFETY DATA SHEET快件声明物安全特性报告MSDS Name（报告物名称）：Monoclonal antibodies Diagnostic RegentSECTION 1---CHEMICAL PRODUCT AND COMPANY IDENTIFICATIO第一部分化工产品及发件单位MSDS Name（报告物名称）：Monoclonal antibodies Diagnostic Regent（单克隆抗体诊断试剂）Catalog Numbers(化工品分类号):N/ASynonyms（又名）：N/ACompany Identification（发件公司名称）：For information, call（一般联系电话）：For emergencies, call（应急联系电话）：SECTION 2---COMPOSITION, INFORMA TION ON INGREDIENTS成分特点Risk Phrases（危险条件）：0SECTION 3---HAZARDS INDENTIFITION危险特征Emergency Overview（概述）Hmis RatingHealth: 0Flammability: 0Reactivity: 0NFPA RatingHealth: 0Flammability: 0Reactivity: 0SECTION 4---FIRST AID MEASURES应急医疗救治Eyes（眼睛）：In case of contact with eyes, flush with copious amounts of water for at least 15minutes. Assure adequate flushing by separating the eyelids with fingers. Call aphysician.Skin（皮肤）：In case of contact, immediately wash skin with soap and copious amounts of water. Ingestion（误食）：If swallowed, wash out mouth with water provided person is conscious. Call a physician.Inhalation（吸入）：If inhaled, remove to fresh air. If breathing becomes difficult, call a physician. Chronic（可否引起慢性病）：N/ASECTION 5---FIRE FIGHTING MEASURES消防扑救方法Extinguishine Media（灭火物质）：Water spray. Carbon dioxide, dry chemical powder, orappropriate foam.Firefighting（防护措施）：Wear self-contained breathing apparatus and protective clothing toprevent contact with skin and eyes.SECTION 6---ACCIDENTAL RELEASE MEASURES事故处理General Information（一般信息）：Exercise appropriate precautions to minimize direct contact with skin or eyes and prevent inhalation of dust.Sweep up, place in a bag and hold for waste disposal.Avoid raising dust.Ventilate area and wash spill site after material pickup is complete.SECTION 7---HANDLING AND STORAGE处理和存放Handling（处理）：Avoid inhalation. Avoid contact with eyes, skin, and clothing. Avoid prolonged or repeated exposure.Storage（存放）：Keep tightly closed.SECTION 8---EXPOSURE CONTROLS, PERSONAL PROTECTION安全控制和人员保护Engineering Control（机械控制）：Safety shower and eye bath. Mechanical exhaust required. Personal Protective Equipment（人员保护）：Use respirators and components tested and approved under appropriate government standards such as NIOSH (US) or CEN (EU). Respiratory protection is not required. Where protection from nuisance levels of dusts are desired, use typeN95 (US) or type P1 (EN 143) dust masks.Hand: Protective gloves.Eye: Chemical safety goggles.General hygiene measures：Wash thoroughly after handling.SECTION 9---PHYSICAL AND CHEMICAL PROPERTIES物理和化学特性Physical State（物理状态）：Liquid Color(颜色)：Red transparentOdor (气味)：Weak Ph: 6.4Vapor Pressure(蒸汽压力)：N/A Viscosity（黏度）：N/ABoiling Point（沸点）：N/A Freezing/Melting Point（冰点）：N/A Autoignition Temperature（自燃温度）：N/A Flash Point（闪点）：N/AExplosion Limits,lower（爆炸极限，低）：N/A Explosion Limits,upper（爆炸极限，高）：N/A Decomposition temperature(分解温度): N/A Solubility in water(溶解度): N/ASpecific Gravity/Density(特定浓度):N/A Molecular Formula(分子式):N/AMolecular Weight(分子量): N/ASECTION 10---STABILITY AND REACTIVTY稳定性和活性Chemical Stability(化学稳定性):StableConditions to Avoid(应避免的环境): Strong oxidizing agents.SECTION 11---TOXICOLOGICAL INFORMA TION毒性信息RTECS#(毒性数据符号): N/ASECTION 12---ECOLOGICAL INFORMATION生态学信息N/ASECTION 13---DISPOSAL CONSIDERATIONS销毁建议Contact a licensed professional waste disposal service to dispose of this material. Dissolve or mix the material with a combustible solvent and burn in a chemical incinerator equipped with an Afterburner and scrubber. Observe all federal, state, and local environmental regulations.SECTION 14---TRANSPORT INFORMA TION空运信息IATA: Non-hazardous for air transport.Reported byCompany Name: 湖南远泰生物技术有限公司Date; 2012-07-28Authorized Person: Jian Qiang Signature: 简强。

镍柱纯化蛋白说明书

Instruction ManualProBond TM Purification SystemFor purification of polyhistidine-containing recombinant proteinsCatalog nos. K850-01, K851-01, K852-01, K853-01, K854-01,R801-01, R801-15Version K2 September200425-0006iiTable of ContentsKit Contents and Storage (iv)Accessory Products (vi)Introduction (1)Overview (1)Methods (2)Preparing Cell Lysates (2)Purification Procedure—Native Conditions (7)Purification Procedure—Denaturing Conditions (11)Purification Procedure—Hybrid Conditions (13)Troubleshooting (15)Appendix (17)Additional Protocols (17)Recipes (18)Frequently Asked Questions (21)References (22)Technical Service (23)iiiKit Contents and StorageTypes of Products This manual is supplied with the following products:Product CatalogNo.ProBond™ Purification System K850-01ProBond™ Purification System with Antibodywith Anti-Xpress™ Antibody K851-01with Anti-myc-HRP Antibody K852-01with Anti-His(C-term)-HRP Antibody K853-01with Anti-V5-HRP Antibody K854-01ProBond™ Nickel-Chelating Resin (50 ml) R801-01ProBond™ Nickel Chelating Resin (150 ml) R801-15ProBond™Purification System Components The ProBond™ Purification System includes enough resin, reagents, and columns for six purifications. The components are listed below. See next page for resin specifications.Component Composition Quantity ProBond™ Resin 50% slurry in 20% ethanol 12 ml5X NativePurification Buffer250 mM NaH2PO4, pH 8.02.5 M NaCl1 × 125 ml bottleGuanidinium LysisBuffer6 M Guanidine HCl20 mM sodium phosphate, pH 7.8500 mM NaCl1 × 60 ml bottleDenaturingBinding Buffer8 M Urea20 mM sodium phosphate, pH 7.8500 mM NaCl2 × 125 ml bottlesDenaturing WashBuffer8 M Urea20 mM sodium phosphate, pH 6.0500 mM NaCl2 × 125 ml bottlesDenaturing ElutionBuffer8 M Urea20 mM NaH2PO4, pH 4.0500 mM NaCl1 × 60 ml bottleImidazole 3 M Imidazole,20 mM sodium phosphate, pH 6.0500 mM NaCl1 × 8 ml bottlePurificationColumns10 ml columns 6Continued on next pageivKit Contents and Storage, ContinuedProBond™Purification System with Antibody The ProBond™ Purification System with Antibody includes resin, reagents, and columns as described for the ProBond™ Purification System (previous page) and 50 µl of the appropriate purified mouse monoclonal antibody. Sufficient reagents are included to perform six purifications and 25 Western blots with the antibody.For more details on the antibody specificity, subclass, and protocols for using the antibody, refer to the antibody manual supplied with the system.Storage Store ProBond™ resin at +4°C. Store buffer and columns at room temperature.Store the antibody at 4°C. Avoid repeated freezing and thawing of theantibody as it may result in loss of activity.The product is guaranteed for 6 months when stored properly.All native purification buffers are prepared from the 5X Native PurificationBuffer and the 3 M Imidazole, as described on page 7.The Denaturing Wash Buffer pH 5.3 is prepared from the Denaturing WashBuffer (pH 6.0), as described on page 11.Resin and ColumnSpecificationsProBond™ resin is precharged with Ni2+ ions and appears blue in color. It isprovided as a 50% slurry in 20% ethanol.ProBond™ resin and purification columns have the following specifications:• Binding capacity of ProBond™ resin: 1–5 mg of protein per ml of resin• Average bead size: 45–165 microns• Pore size of purification columns: 30–35 microns• Recommended flow rate: 0.5 ml/min• Maximum flow rate: 2 ml/min• Maximum linear flow rate: 700 cm/h• Column material: Polypropylene• pH stability (long term): pH 3–13• pH stability (short term): pH 2–14ProductQualificationThe ProBond™ Purification System is qualified by purifying 2 mg of myoglobinprotein on a column and performing a Bradford assay. Protein recovery mustbe 75% or higher.vAccessory ProductsAdditionalProductsThe following products are also available for order from Invitrogen:Product QuantityCatalogNo.ProBond™ Nickel-Chelating Resin 50 ml150 mlR801-01R801-15Polypropylene columns(empty)50 R640-50Ni-NTA Agarose 10 ml25 ml R901-01 R901-15Ni-NTA Purification System 6 purifications K950-01 Ni-NTA Purification Systemwith Antibodywith Anti-Xpress™ Antibody with Anti-myc-HRP Antibody with Anti-His(C-term)-HRP Antibodywith Anti-V5-HRP Antibody 1 kit1 kit1 kit1 kitK951-01K952-01K953-01K954-01Anti-myc Antibody 50 µl R950-25 Anti-V5 Antibody 50 µl R960-25 Anti-Xpress™ Antibody 50 µl R910-25 Anti-His(C-term) Antibody 50 µl R930-25 InVision™ His-tag In-gel Stain 500 ml LC6030 InVision™ His-tag In-gelStaining Kit1 kit LC6033Pre-Cast Gels and Pre-made Buffers A large variety of pre-cast gels for SDS-PAGE and pre-made buffers for your convenience are available from Invitrogen. For details, visit our web site at or contact Technical Service (page 23).viIntroductionOverviewIntroduction The ProBond™ Purification System is designed for purification of 6xHis-tagged recombinant proteins expressed in bacteria, insect, and mammalian cells. Thesystem is designed around the high affinity and selectivity of ProBond™Nickel-Chelating Resin for recombinant fusion proteins containing six tandemhistidine residues.The ProBond™ Purification System is a complete system that includespurification buffers and resin for purifying proteins under native, denaturing,or hybrid conditions. The resulting proteins are ready for use in many targetapplications.This manual is designed to provide generic protocols that can be adapted foryour particular proteins. The optimal purification parameters will vary witheach protein being purified.ProBond™ Nickel-Chelating Resin ProBond™ Nickel-Chelating Resin is used for purification of recombinant proteins expressed in bacteria, insect, and mammalian cells from any 6xHis-tagged vector. ProBond™ Nickel-Chelating Resin exhibits high affinity and selectivity for 6xHis-tagged recombinant fusion proteins.Proteins can be purified under native, denaturing, or hybrid conditions using the ProBond™ Nickel-Chelating Resin. Proteins bound to the resin are eluted with low pH buffer or by competition with imidazole or histidine. The resulting proteins are ready for use in target applications.Binding Characteristics ProBond™ Nickel-Chelating Resin uses the chelating ligand iminodiacetic acid (IDA) in a highly cross-linked agarose matrix. IDA binds Ni2+ ions by three coordination sites.The protocols provided in this manual are generic, and may not result in 100%pure protein. These protocols should be optimized based on the bindingcharacteristics of your particular proteins.Native VersusDenaturingConditionsThe decision to purify your 6xHis-tagged fusion proteins under native ordenaturing conditions depends on the solubility of the protein and the need toretain biological activity for downstream applications.• Use native conditions if your protein is soluble (in the supernatant afterlysis) and you want to preserve protein activity.• Use denaturing conditions if the protein is insoluble (in the pellet afterlysis) or if your downstream application does not depend on proteinactivity.• Use hybrid protocol if your protein is insoluble but you want to preserveprotein activity. Using this protocol, you prepare the lysate and columnsunder denaturing conditions and then use native buffers during the washand elution steps to refold the protein. Note that this protocol may notrestore activity for all proteins. See page 14.1MethodsPreparing Cell LysatesIntroduction Instructions for preparing lysates from bacteria, insect, and mammalian cellsusing native or denaturing conditions are described below.Materials Needed You will need the following items:• Native Binding Buffer (recipe is on page 8) for preparing lysates undernative conditions• Sonicator• 10 µg/ml RNase and 5 µg/ml DNase I (optional)• Guanidinium Lysis Buffer (supplied with the system) for preparing lysatesunder denaturing conditions• 18-gauge needle• Centrifuge• Sterile, distilled water• SDS-PAGE sample buffer• Lysozyme for preparing bacterial cell lysates• Bestatin or Leupeptin, for preparing mammalian cell lysatesProcessing Higher Amount of Starting Material Instructions for preparing lysates from specific amount of starting material (bacteria, insect, and mammalian cells) and purification with 2 ml resin under native or denaturing conditions are described in this manual.If you wish to purify your protein of interest from higher amounts of starting material, you may need to optimize the lysis protocol and purification conditions (amount of resin used for binding). The optimization depends on the expected yield of your protein and amount of resin to use for purification. Perform a pilot experiment to optimize the purification conditions and then based on the pilot experiment results, scale-up accordingly.Continued on next page2Preparing Bacterial Cell Lysate—Native Conditions Follow the procedure below to prepare bacterial cell lysate under native conditions. Scale up or down as necessary.1. Harvest cells from a 50 ml culture by centrifugation (e.g., 5000 rpm for5 minutes in a Sorvall SS-34 rotor). Resuspend the cells in 8 ml NativeBinding Buffer (recipe on page 8).2. Add 8 mg lysozyme and incubate on ice for 30 minutes.3. Using a sonicator equipped with a microtip, sonicate the solution on iceusing six 10-second bursts at high intensity with a 10-second coolingperiod between each burst.Alternatively, sonicate the solution on ice using two or three 10-secondbursts at medium intensity, then flash freeze the lysate in liquid nitrogen or a methanol dry ice slurry. Quickly thaw the lysate at 37°C andperform two more rapid sonicate-freeze-thaw cycles.4. Optional: If the lysate is very viscous, add RNase A (10 µg/ml) andDNase I (5 µg/ml) and incubate on ice for 10–15 minutes. Alternatively,draw the lysate through a 18-gauge syringe needle several times.5. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris. Transfer the supernatant to a fresh tube.Note: Some 6xHis-tagged protein may remain insoluble in the pellet, and can be recovered by preparing a denatured lysate (page 4) followed bythe denaturing purification protocol (page 12). To recover this insolubleprotein while preserving its biological activity, you can prepare thedenatured lysate and then follow the hybrid protocol on page 14. Notethat the hybrid protocol may not restore activity in all cases, and should be tested with your particular protein.6. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaininglysate on ice or freeze at -20°C. When ready to use, proceed to theprotocol on page 7.Continued on next page3Preparing Bacterial Cell Lysate—Denaturing Conditions Follow the procedure below to prepare bacterial cell lysate under denaturing conditions:1. Equilibrate the Guanidinium Lysis Buffer, pH 7.8 (supplied with thesystem or see page 19 for recipe) to 37°C.2. Harvest cells from a 50 ml culture by centrifugation (e.g., 5000 rpm for5 minutes in a Sorvall SS-34 rotor).3. Resuspend the cell pellet in 8 ml Guanidinium Lysis Buffer from Step 1.4. Slowly rock the cells for 5–10 minutes at room temperature to ensurethorough cell lysis.5. Sonicate the cell lysate on ice with three 5-second pulses at high intensity.6. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris.Transfer the supernatant to a fresh tube.7. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaininglysate on ice or at -20°C. When ready to use, proceed to the denaturingprotocol on page 11 or hybrid protocol on page 13.Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer, you need to dilute the samples, dialyze the samples, or perform TCAprecipitation prior to SDS-PAGE to prevent the precipitation of SDS.Harvesting Insect Cells For detailed protocols dealing with insect cell expression, consult the manual for your particular system. The following lysate protocols are for baculovirus-infected cells and are intended to be highly generic. They should be optimized for your cell lines.For baculovirus-infected insect cells, when the time point of maximal expression has been determined, large scale protein expression can be carried out. Generally, the large-scale expression is performed in 1 liter flasks seeded with cells at a density of 2 × 106 cells/ml in a total volume of 500 ml and infected with high titer viral stock at an MOI of 10 pfu/cell. At the point of maximal expression, harvest cells in 50 ml aliquots. Pellet the cells by centrifugation and store at -70°C until needed. Proceed to preparing cell lysates using native or denaturing conditions as described on the next page.Continued on next page4Preparing Insect Cell Lysate—Native Condition 1. Prepare 8 ml Native Binding Buffer (recipe on page 8) containingLeupeptin (a protease inhibitor) at a concentration of 0.5 µg/ml.2. After harvesting the cells (previous page), resuspend the cell pellet in8 ml Native Binding Buffer containing 0.5 µg/ml Leupeptin.3. Lyse the cells by two freeze-thaw cycles using a liquid nitrogen or dryice/ethanol bath and a 42°C water bath.4. Shear DNA by passing the preparation through an 18-gauge needle fourtimes.5. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris.Transfer the supernatant to a fresh tube.6. Remove 5 µl of the lysate for SDS-PAGE analysis. Store remaining lysateon ice or freeze at -20°C. When ready to use, proceed to the protocol on page 7.Preparing Insect Cell Lysate—Denaturing Condition 1. After harvesting insect cells (previous page), resuspend the cell pellet in8 ml Guanidinium Lysis Buffer (supplied with the system or see page 19for recipe).2. Pass the preparation through an 18-gauge needle four times.3. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris. Transfer the supernatant to a fresh tube.4. Remove 5 µl of the lysate for SDS-PAGE analysis. Store remaining lysateon ice or freeze at -20° C. When ready to use, proceed to the denaturingprotocol on page 11 or hybrid protocol on page 13.Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer, you need to dilute the samples, dialyze the samples, or perform TCAprecipitation prior to SDS-PAGE to prevent the precipitation of SDS.Continued on next pagePreparing Mammalian Cell Lysate—Native Conditions For detailed protocols dealing with mammalian expression, consult the manual for your particular system. The following protocols are intended to be highly generic, and should be optimized for your cell lines.To produce recombinant protein, you need between 5 x 106and 1 x 107 cells. Seed cells and grow in the appropriate medium until they are 80–90% confluent. Harvest cells by trypsinization. You can freeze the cell pellet in liquid nitrogen and store at -70°C until use.1. Resuspend the cell pellet in 8 ml of Native Binding Buffer (page 8). Theaddition of protease inhibitors such as bestatin and leupeptin may benecessary depending on the cell line and expressed protein.2. Lyse the cells by two freeze-thaw cycles using a liquid nitrogen or dryice/ethanol bath and a 42°C water bath.3. Shear the DNA by passing the preparation through an 18-gauge needlefour times.4. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris. Transfer the supernatant to a fresh tube.5. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaininglysate on ice or freeze at -20° C. When ready to use, proceed to theprotocol on page 7.Preparing Mammalian Cell Lysates—Denaturing Conditions For detailed protocols dealing with mammalian expression, consult the manual for your particular system. The following protocols are intended to be highly generic, and should be optimized for your cell lines.To produce recombinant protein, you need between 5 x 106and 1 x 107 cells. Seed cells and grow in the appropriate medium until they are 80–90% confluent. Harvest cells by trypsinization. You can freeze the cell pellet in liquid nitrogen and store at -70°C until use.1. Resuspend the cell pellet in 8 ml Guanidinium Lysis Buffer (suppliedwith the system or see page 19 for recipe).2. Shear the DNA by passing the preparation through an 18-gauge needlefour times.3. Centrifuge the lysate at 3,000 ×g for 15 minutes to pellet the cellulardebris. Transfer the supernatant to a fresh tube.4. Remove 5 µl of the lysate for SDS-PAGE analysis. Store the remaininglysate on ice or freeze at -20° C until use. When ready to use, proceed to the denaturing protocol on page 11 or hybrid protocol on page 13.Note: To perform SDS-PAGE with samples in Guanidinium Lysis Buffer, you need to dilute the samples, dialyze the samples, or perform TCAprecipitation prior to SDS-PAGE to prevent the precipitation of SDS.Purification Procedure—Native ConditionsIntroduction In the following procedure, use the prepared Native Binding Buffer, NativeWash Buffer, and Native Elution Buffer, columns, and cell lysate preparedunder native conditions. Be sure to check the pH of your buffers before starting.Buffers for Native Purification All buffers for purification under native conditions are prepared from the5X Native Purification Buffer supplied with the system. Dilute and adjust the pH of the 5X Native Purification Buffer to create 1X Native Purification Buffer (page 8). From this, you can create the following buffers:• Native Binding Buffer• Native Wash Buffer• Native Elution BufferThe recipes described in this section will create sufficient buffers to perform one native purification using one kit-supplied purification column. Scale up accordingly.If you are preparing your own buffers, see page 18 for recipe.Materials Needed You will need the following items:• 5X Native Purification Buffer (supplied with the system or see page 18 forrecipe)• 3 M Imidazole (supplied with the system or see page 18 for recipe)• NaOH• HCl• Sterile distilled water• Prepared ProBond™ columns with native buffers (next page)• Lysate prepared under native conditions (page 2)Imidazole Concentration in Native Buffers Imidazole is included in the Native Wash and Elution Buffers to minimize the binding of untagged, contaminating proteins and increase the purity of the target protein with fewer wash steps. Note that, if your level of contaminating proteins is high, you may add imidazole to the Native Binding Buffer.If your protein does not bind well under these conditions, you can experiment with lowering or eliminating the imidazole in the buffers and increasing the number of wash and elution steps.Continued on next page1X Native Purification Buffer To prepare 100 ml 1X Native Purification Buffer, combine:• 80 ml of sterile distilled water• 20 ml of 5X Native Purification Buffer (supplied with the system or see page 18 for recipe)Mix well and adjust pH to 8.0 with NaOH or HCl.Native Binding Buffer Without ImidazoleUse 30 ml of the 1X Native Purification Buffer (see above for recipe) for use as the Native Binding Buffer (used for column preparation, cell lysis, and binding).With Imidazole (Optional):You can prepare the Native Binding Buffer with imidazole to reduce the binding of contaminating proteins. (Note that some His-tagged proteins may not bind under these conditions.).To prepare 30 ml Native Binding Buffer with 10 mM imidazole, combine: • 30 ml of 1X Native Purification Buffer• 100 µl of 3 M Imidazole, pH 6.0Mix well and adjust pH to 8.0 with NaOH or HCl.Native Wash Buffer To prepare 50 ml Native Wash Buffer with 20 mM imidazole, combine:• 50 ml of 1X Native Purification Buffer• 335 µl of 3 M Imidazole, pH 6.0Mix well and adjust pH to 8.0 with NaOH or HCl.Native Elution Buffer To prepare 15 ml Native Elution Buffer with 250 mM imidazole, combine:• 13.75 ml of 1X Native Purification Buffer• 1.25 ml of 3 M Imidazole, pH 6.0Mix well and adjust pH to 8.0 with NaOH or HCl.Continued on next pageDo not use strong reducing agents such as DTT with ProBond™ columns. DTTreduces the nickel ions in the resin. In addition, do not use strong chelatingagents such as EDTA or EGTA in the loading buffers or wash buffers, as thesewill strip the nickel from the columns.Be sure to check the pH of your buffers before starting.PreparingProBond™ ColumnWhen preparing a column as described below, make sure that the snap-off capat the bottom of the column remains intact. To prepare a column:1. Resuspend the ProBond™ resin in its bottle by inverting and gentlytapping the bottle repeatedly.2. Pipet or pour 2 ml of the resin into a 10-ml Purification Columnsupplied with the kit. Allow the resin to settle completely by gravity(5-10 minutes) or gently pellet it by low-speed centrifugation (1 minuteat 800 ×g). Gently aspirate the supernatant.3. Add 6 ml of sterile, distilled water and resuspend the resin byalternately inverting and gently tapping the column.4. Allow the resin to settle using gravity or centrifugation as described inStep 2, and gently aspirate the supernatant.5. For purification under Native Conditions, add 6 ml Native BindingBuffer (recipe on page 8).6. Resuspend the resin by alternately inverting and gently tapping thecolumn.7. Allow the resin to settle using gravity or centrifugation as described inStep 2, and gently aspirate the supernatant.8. Repeat Steps 5 through 7.Storing PreparedColumnsTo store a column containing resin, add 0.02% azide or 20% ethanol as apreservative and cap or parafilm the column. Store at room temperature.Continued on next pagePurification Under Native Conditions Using the native buffers, columns and cell lysate, follow the procedure below to purify proteins under native conditions:1. Add 8 ml of lysate prepared under native conditions to a preparedPurification Column (page 9).2. Bind for 30–60 minutes using gentle agitation to keep the resinsuspended in the lysate solution.3. Settle the resin by gravity or low speed centrifugation (800 ×g), andcarefully aspirate the supernatant. Save supernatant at 4°C forSDS-PAGE analysis.4. Wash with 8 ml Native Wash Buffer (page 8). Settle the resin by gravityor low speed centrifugation (800 ×g), and carefully aspirate thesupernatant. Save supernatant at 4°C for SDS-PAGE analysis.5. Repeat Step 4 three more times.6. Clamp the column in a vertical position and snap off the cap on thelower end. Elute the protein with 8–12 ml Native Elution Buffer (seepage 2). Collect 1 ml fractions and analyze with SDS-PAGE.Note: Store the eluted fractions at 4°C. If -20°C storage is required, addglycerol to the fractions. For long term storage, add protease inhibitors to the fractions.If you wish to reuse the resin to purify the same recombinant protein, wash the resin with 0.5 M NaOH for 30 minutes and equilibrate the resin in a suitable binding buffer. If you need to recharge the resin, see page 17.Purification Procedure—Denaturing ConditionsIntroduction Instructions to perform purification using denaturing conditions with prepareddenaturing buffers, columns, and cell lysate are described below.Materials Needed You will need the following items:• Denaturing Binding Buffer (supplied with the system or see page 19 forrecipe)• Denaturing Wash Buffer, pH 6.0 (supplied with the system or see page 19 forrecipe) and Denaturing Wash Buffer, pH 5.3 (see recipe below)• Denaturing Elution Buffer (supplied with the system or see page 20 forrecipe)• Prepared ProBond™ columns with Denaturing buffers (see below)• Lysate prepared under denaturing conditions (page 11)Preparing the Denaturing Wash Buffer pH 5.3 Using a 10 ml aliquot of the kit-supplied Denaturing Wash Buffer (pH 6.0), mix well, and adjust the pH to 5.3 using HCl. Use this for the Denaturing Wash Buffer pH 5.3 in Step 5 next page.Be sure to check the pH of your buffers before starting. Note that thedenaturing buffers containing urea will become more basic over time. PreparingProBond™ ColumnWhen preparing a column as described below, make sure that the snap-off capat the bottom of the column remains intact.If you are reusing the ProBond™ resin, see page 17 for recharging protocol.To prepare a column:1. Resuspend the ProBond™ resin in its bottle by inverting and gentlytapping the bottle repeatedly.2. Pipet or pour 2 ml of the resin into a 10-ml Purification Columnsupplied with the kit. Allow the resin to settle completely by gravity(5-10 minutes) or gently pellet it by low-speed centrifugation (1 minuteat 800 ×g). Gently aspirate the supernatant.3. Add 6 ml of sterile, distilled water and resuspend the resin byalternately inverting and gently tapping the column.4. Allow the resin to settle using gravity or centrifugation as described inStep 2, and gently aspirate the supernatant.5. For purification under Denaturing Conditions, add 6 ml of DenaturingBinding Buffer.6. Resuspend the resin by alternately inverting and gently tapping thecolumn.7. Allow the resin to settle using gravity or centrifugation as described inStep 2, and gently aspirate the supernatant. Repeat Steps 5 through 7.Continued on next pagePurification Procedure—Denaturing Conditions, ContinuedPurification Under Denaturing Conditions Using the denaturing buffers, columns, and cell lysate, follow the procedure below to purify proteins under denaturing conditions:1. Add 8 ml lysate prepared under denaturing conditions to a preparedPurification Column (page 11).2. Bind for 15–30 minutes at room temperature using gentle agitation (e.g.,using a rotating wheel) to keep the resin suspended in the lysatesolution. Settle the resin by gravity or low speed centrifugation (800 ×g), and carefully aspirate the supernatant.3. Wash the column with 4 ml Denaturing Binding Buffer supplied with thekit by resuspending the resin and rocking for two minutes. Settle theresin by gravity or low speed centrifugation (800 ×g), and carefullyaspirate the supernatant. Save supernatant at 4°C for SDS-PAGEanalysis. Repeat this step one more time.4. Wash the column with 4 ml Denaturing Wash Buffer, pH 6.0 supplied inthe kit by resuspending the resin and rocking for two minutes. Settle the resin by gravity or low speed centrifugation (800 ×g), and carefullyaspirate the supernatant. Save supernatant at 4°C for SDS-PAGEanalysis. Repeat this step one more time.5. Wash the column with 4 ml Denaturing Wash Buffer pH 5.3 (see recipeon previous page) by resuspending the resin and rocking for 2 minutes.Settle the resin by gravity or low speed centrifugation (800 ×g), andcarefully aspirate the supernatant. Save supernatant at 4°C for SDS-PAGE analysis. Repeat this step once more for a total of two washes with Denaturing Wash Buffer pH 5.3.6. Clamp the column in a vertical position and snap off the cap on thelower end. Elute the protein by adding 5 ml Denaturing Elution Buffersupplied with the kit. Collect 1 ml fractions and monitor the elution bytaking OD280readings of the fractions. Pool the fractions that contain the peak absorbance and dialyze against 10 mM Tris, pH 8.0, 0.1% Triton X-100 overnight at 4°C to remove the urea. Concentrate the dialyzedmaterial by any standard method (i.e., using 10,000 MW cut-off, low-protein binding centrifugal instruments or vacuum concentrationinstruments).If you wish to reuse the resin to purify the same recombinant protein, wash the resin with 0.5 M NaOH for 30 minutes and equilibrate the resin in a suitable binding buffer. If you need to recharge the resin, see page 17.。

西北大学细胞生物学

西北大学2002年细胞生物学试题一.名词解释。

（20分）1.有丝分裂（mitosis）2.印迹技术（blotting）3.细胞膜（cell membrane）4.单克隆抗体技术（monoclonal antiboday）5.胞质体（cytoplast）6.第二信使（secondary messenger）7.载体8.微粒体(microsomes)9.核基质（nuclear matrix）10.干细胞（stem cell）二.比较真核细胞和原核细胞。

10分三.细胞质骨架有哪些成分？试比较它们的主要特征。

10分四.真核细胞的蛋白质合成有什么特点？20分五.染色体和染色质有什么区别？染色体要确保在世代中的稳定性起码要具备哪些结构要素？染色体是怎样包装形成的？20分六.详细分析细胞周期调控机制。

20分西北大学2003年招收攻读硕士研究生试题科目名称：细胞生物学科目代码：340适用专业：细胞生物学一.名词解释（每词3分共60分）1.原生质体2.逆转病毒3.真细菌4.质粒5.细胞外被6.反式作用因子7.人造微小染色体8.溶酶体9.G蛋白10.蛋白质免疫印迹技术11.胞质体12.GFP13.生物膜14.微粒体15.多聚核糖体16.Northern 杂交17.信号肽18.同源染色体19.旁分泌20.巴尔氏小体二.物质通过细胞膜的转运途径有哪些？试举例说明。

10分三.分析内质网的类型及其功能。

10分四.试分析叶绿体形态结构与功能的关系。

10分五.分析中期染色体的形态结构和染色体DNA的三种功能元件，比较染色质包装的多级螺旋模型和骨架-放射环模型。

20分六.细胞周期调控的分子机制是什么？20分七.广义的细胞骨架包括哪些成分，它们的结构特征和功能是什么？20分西北大学2004年招收攻读硕士研究生试题科目名称：细胞生物学科目代码：330一.名词解释（每词3分共60分）1.选择素2.第二信使3.内膜系统4.微粒体5.短散在重复元件6.DNA指纹技术7.姐妹染色单体8.Y AC9.TUNEL测定法10.基因组11.信使RNA12.真细菌13.Northern印迹杂交技术14.酵母双杂交技术15.圆球体16.连接子17.分辨率18.生物信息学19.外显子20.蛋白酶体二.分析逆转录病毒的侵染机理及其在基因治疗中的应用价值。

制备多克隆抗体的流程

制备多克隆抗体的流程英文回答：The process of generating monoclonal antibodies involves several steps. Here is a general outline of the procedure:1. Immunization: The first step is to immunize an animal, typically a mouse or a rat, with the specific antigen of interest. The antigen can be a protein, peptide, or even a whole cell. The animal's immune system recognizes the antigen as foreign and mounts an immune response, producing a diverse population of antibodies.2. Cell Fusion: After a sufficient immune response is generated, the next step is to harvest immune cells, usually from the spleen, of the immunized animal. These cells, called B cells, are responsible for producing antibodies. B cells are fused with myeloma cells, a type of cancerous cell line that can divide indefinitely. Thisfusion creates hybridoma cells, which have the ability to produce antibodies and divide indefinitely.3. Selection: The fused cells are then cultured in a selective medium that allows only the hybridoma cells to survive. This medium usually contains a substance that prevents the growth of unfused cells and myeloma cells. The surviving hybridoma cells are then screened for antibody production.4. Screening: Screening involves testing the culture supernatants of the hybridoma cells for the presence of specific antibodies against the antigen of interest. Various techniques can be used for screening, such as enzyme-linked immunosorbent assay (ELISA) or immunofluorescence. Positive clones that produce the desired antibodies are selected for further analysis.5. Cloning: To ensure monoclonality, the selected hybridoma cells are subjected to limiting dilution, where single cells are distributed into individual wells of a culture plate. This process allows for the isolation ofindividual clones derived from a single cell. Each clone is expanded and tested for antibody production.6. Antibody Production: The selected monoclonal antibody-producing clones are grown in culture to produce a large quantity of antibodies. The antibodies can be harvested from the culture supernatant or purified using various techniques, such as protein A/G affinity chromatography.7. Characterization: The generated monoclonal antibodies are characterized for their specificity, affinity, and functionality. This involves further testing, such as Western blotting, immunohistochemistry, or flow cytometry, to determine the antibody's ability to recognize the target antigen.8. Scale-up and Production: Once the monoclonal antibodies are characterized, they can be scaled up for production. This involves large-scale culture of the selected hybridoma cells to generate a substantial amount of antibodies for research or therapeutic applications.中文回答：制备多克隆抗体的流程涉及几个步骤。

Spitz痣一例并相关文献复习

Spitz痣一例并相关文献复习患儿，女性，5岁8月，右侧面颊部5年前发现小米粒样褐色丘疹，始无疼痛、瘙痒，未行处理，近一年明显增大，无自觉症状，因影响外观并怀疑恶变，来我院就诊。

专科检查：右侧面颊部约0.4cm×0.4cm隆起包块，红褐色，孤立存在，无毛发生长，无压痛，无破溃。

临床处理：适当扩大切除病变组织。

病理检查结果考虑为Spitz痣（上皮样细胞型），免疫组织化学示：melant(+)，S-100(+)，HMB-45局灶少数细胞（+），ki-67阳性率约为2%（图二）。

讨论Spitz痣又名良性幼年黑色素瘤或梭形细胞痣、上皮样细胞痣，是混合痣的一种异性，常见于儿童，也可发生于成人[1]。

由Sophie Spitz于1948年首先报道，并命名。

经典的临床表现为因缺乏黑色素而形成淡粉红色半球形丘疹，部分呈红褐色、褐色、炭黑色[2]。

大多数<1cm，一般常见单发，少数多发[3]。

Spitz痣的细胞体积较大，有梭形细胞和上皮样细胞，约半数以上的病例以梭形细胞为主，20%病例以上皮细胞为主。

本例几乎多数为上皮样细胞。

临床上需要与血管瘤、化脓性肉芽肿、皮肤纤维瘤、黑色素细胞瘤鉴别。

Spitz痣在形态学、组织病理上与黑色素瘤十分相似，因此有人称为摹拟黑色素瘤[4]，其他病变依据形态特点及免疫组化不难鉴别。

有研究指出约6.5%Spitz痣误诊为恶性黑色素瘤，超过8%的病例组织病理学与恶性黑色素瘤难以区别。

所以Spitz痣与恶性黑色素瘤的鉴别诊断尤为重要。

①Spitz痣多见于儿童，界限清楚，基底痣细胞成熟，可见群集的kamino小体；黑色素细胞瘤多见于成人，边界不清，肿瘤细胞多不具备成熟现象（趋神经分化型除外），少见kamino小体。

[5][6]②黑素瘤常有表皮内瘤细胞浸润和基底层单个黑素细胞增生；痣细胞位于真皮浅层和深层。

③此外免疫组化技术在临床上对于二者的鉴别具有重要意义：Spitz痣HMB-45抗原在真皮表浅部位表达；黑素瘤真皮全层均表达为阳性。

EV71病毒特异性单克隆抗体的筛选

分类号：单位代码：10019密级：学号：TS040322硕士学位论文EV71病毒特异性单克隆抗体的筛选与鉴定研究Screening and Characterization of Monoclonal Antibody Specific to EV71 Virus研究生：李静指导教师：王宾教授申请学位门类级别：理学硕士专业名称：生理学研究方向：分子免疫学所在学院：生物学院二零零九年六月独创性声明本人声明所呈交的论文是我个人在导师指导下进行的研究工作及取得的研究成果。

尽我所知，除了文中特别加以标注和致谢的地方外，论文中不包含其他人已经发表或撰写过的研究成果，也不包含为获得中国农业大学或其它教育机构的学位或证书而使用过的材料。

与我一同工作的同志对本研究所做的任何贡献均已在论文中作了明确的说明并表示了谢意。

研究生签名：时间：年月日关于论文使用授权的说明本人完全了解中国农业大学有关保留、使用学位论文的规定，即：学校有权保留送交论文的复印件和磁盘，允许论文被查阅和借阅，可以采用影印、缩印或扫描等复制手段保存、汇编学位论文。

同意中国农业大学可以用不同方式在不同媒体上发表、传播学位论文的全部或部分内容。

(保密的学位论文在解密后应遵守此协议)研究生签名：时间：年月日导师签名：时间：年月日摘要手足口病（Hand foot mouth disease， HFMD）是由多种肠道病毒引起的一种常见急性传染病，多发生于5岁以下的婴幼儿，可引起发热和手足、口腔等部位的皮疹、溃疡，个别患者可引起心肌炎、肺水肿、无菌性脑膜脑炎等致命性并发症。

该病是全球性传染病，传染性强，易引起暴发或流行。

1997年以来，肠道病毒71型（Enterovirus 71， EV71）感染为主的手足口病在马来西亚、台湾、新加坡等地大规模爆发流行，引起世界各国关注和警惕。

EV71病毒为小RNA病毒科、肠道病毒属，分为A、B、C三基因型；VP1作为主要的衣壳蛋白，具有最多的型特异性中和位点，是主要的病毒中和决定因子，直接决定病毒的抗原性。

《生物技术药物》之抗体、疫苗与佐剂

抗体(单克隆抗体)

脾脏有上百万B淋巴细胞，具有合成不同的抗体能力。当机体受抗原刺激时，抗原分子上的许多决定簇分别激活各个具有不同基因的B细胞。被激活的B细胞分裂增殖形成效应B细胞（浆细胞）和记忆B细胞，大量的浆细胞克隆合成和分泌大量的抗体分子分布到血液、体液中。如果能选出一个制造一种专一抗体的浆细胞进行培养，就可得到由单细胞经分裂增殖而形成细胞群，即单克隆。单克隆细胞将合成针对一种抗原决定簇的抗体，称为单克隆抗体。

After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. The contaminants in the cell culture sample would consist primarily of media components such as growth factors, hormones, and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic acids, and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as a result, endotoxins that are secreted by the bacteria. Depending on the complexity of the media required in cell culture, and thus the contaminants in question, one method (in vivo or in vitro) may be preferable to the other.

细胞角蛋白(广谱)抗体试剂(免疫组织化学)说明书

【包装规格】
12mL
【预期用途】
体外诊断用途。在常规染色（如：HE染色）基础上进行免疫组织化学染色，为医师提供诊断的辅助信息。细胞角蛋白（广谱）抗体试剂（免疫组织化学），用于在 Dako Omnis 仪器上进行的免疫组化分析。该抗体有助于判定肿瘤是否为恶性或者上皮来源 (1-3)。临床判定任何染色的着色或缺失都必须有恰当对照的形态学结果作为补充，基于患者的临床病史和其它诊断结果，由有资质的病理医生诊断。
3. Moll R, Franke WW, Schiller Dl. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 1982; 31:11
2. Woodcock-Mitchell J, Eichner R, Nelson WG, Sun TT. Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies. J Cell Biol 1982; 95:580
快速指南
步骤
备注
固定/包埋预处理抗体阴性对照显色
复染对照组织
福尔马林固定，石蜡包埋
机内脱蜡
EnVision™ FLEX，高pH（编码GV804）热诱导的抗原修复30分钟
即用型
孵育12.5分钟
FLEX阴性对照，小鼠（编码GA750）孵育12.5分钟
EnVision™ FLEX（编码GV800）
*用户必须阅读说明书，以了解染色操作和产品处理的详细说明。

SCI论文写作--方法部分

Insulin resistance
Insulinogenic index
Homeostasis model assessment MPO activity
formula: increment of plasma insulin (0-5min) / increment of plasma glucose (0-5 min) (Kosa et al., 1974).
Neutrophil sequestration in the pancreas was quantified by measuring tissue MPO activity (Bhatia et al., 2005). The suspension was subjected to four cycles of freezing and thawing, and was further disrupted by sonication.
formula: fasting insulin (mU· -1 mL ) x fasting glucose (mM)/22.5 (Matthews et al.,1985).
Neutrophil sequestration 中性粒细胞分离 in the pancreas
Anti· (TGF)-β1 inflammatory · (TNF)-α mRNA and anti-fibrotic · value CT effects of pravastatin
For all types of paper, the Methods section should include sufficient detail and references to permit a trained scientist to evaluate your work fully or to repeat the experiments exactly as you did them.

巨噬细胞吞噬凋亡中性粒细胞后吞噬减少

Previous Uptake of Apoptotic Neutrophils or Ligation of Integrin Receptors Downmodulates the Ability of Macrophages to Ingest Apoptotic Neutrophils By Lars-Peter Erwig,Sharon Gordon,Garry M.Walsh,and Andrew J.ReesClearance of apoptotic neutrophils(polymorphonuclear leu-kocyte[PMN])by macrophages is thought to play a crucial role in resolution of acute inflammation.There is increasing evidence that ingestion of apoptotic cells modulates macro-phage behavior.We therefore performed experiments to determine whether ingestion of apoptotic PMN modulated the uptake process itself.Rat bone marrow-derived macro-phages(BMDM)ingested apoptotic PMN by a process that was enhanced by tumor necrosis factor(TNF)and attenu-ated by interferon(IFN)-␥,interleukin(IL)-4,and IL-10.It was inhibitable by the tetrapeptide arg-gly-gln-ser(RGDS),there-fore implicating the␣v␤3/CD36/thrombospondin pathway. Interaction of apoptotic PMN with BMDM for30minutes,48 hours before rechallenge reduced uptake of apoptotic PMN by50%compared with previously unchallenged BMDM. Blocking initial uptake with RGDS abrogated the effect of parable and sustained attenuation of up-take was obtained by ligating␣v␤3with the monoclonal antibody(MoAb),F11,after a delay of more than90minutes, whereas MoAbs to CD25and CD45had no effect.Ligation of ␣6␤1and␣1␤2,integrins not previously implicated in the engulfment of apoptotic cells also decreased uptake with similar kinetics to F11.Therefore,apoptotic PMN regulate their own uptake through an integrin-dependent process, which can be reproduced by ligation of other integrins expressed by macrophages.௠1999by The American Society of Hematology.M ACROPHAGES INFLUENCE almost all aspects of immunological and inﬂammatory responses and play an essential role in linking the innate and acquired immunity.1 Macrophages not only induce injury,but also control key events in the resolution of inﬂammation and the repair processes that follow it.One of the critical functions in this process is phagocytosis of apoptotic cells via speciﬁc recognition mecha-nisms.To date,a number of recognition mechanisms for apoptotic cells have been described:(1)an uncharacterized lectin-dependent interaction2;(2)a complicated charge sensi-tive process involving the CD36/vitronectin receptor(␣v␤3) complex on the macrophage surface interacting with unknown moieties on the apoptotic polymorphonuclear leukocyte(PMN) surface via a thrombospondin bridge3,4;(3)a stereo-speciﬁc recognition of phosphatidylserine that is expressed on the surface of the apoptotic cell after loss of membrane asymme-try5,6;(4)macrophage scavenger receptors7;(5)the lipopolysac-charide(LPS)receptor CD148-10and macrosialin or CD68.11,12 The speciﬁc removal of apoptotic thymocytes,13eosino-phils,14and neutrophils15by macrophages has been well described.Extensive tissue damage and inﬂammation both precede and follow neutrophil death by necrosis.The cellular debris is phagocytosed by macrophages,which are activated by the process.In contrast,apoptosis of neutrophils is associated with the swift recognition of intact cells by macrophages followed by their ingestion and degradation.Local inﬂamma-tion and tissue injury are avoided not only because neutrophils are prevented from releasing their toxic contents,but also because the macrophages usual proinﬂammatory secretory response to phagocytosis is not activated16and may be biased towards release of the anti-inﬂammatory cytokine transforming growth factor(TGF)-␤.17These results suggest that uptake of apoptotic neutrophils by macrophages does not merely fail to induce synthesis of proinﬂammatory cytokines,but actively modulates macrophage function and biases the proﬁle of cytokines they release.This raises the question whether uptake of apoptotic cells‘‘imprints’’a pattern of behavior on macrophages analogous to the effect of exposure to some cytokines.18The speciﬁc purpose of the experiments described here was to ascertain whether uptake of apoptotic neutrophils modulates the ability of macrophages to ingest a second challenge with apoptotic PMNs.The results show a substantial reduction in the proportion of macrophages that ingest a second challenge of apoptotic PMNs,but that uptake can still be modulated appropriately by cytokines.The bone marrow-derived macrophages(BMDM)uptake of apop-totic PMN is RGDS-dependent and presumptively occurs by the ␣v␤3/CD36/thrombospondin pathway.After a delay of at least 90minutes,ligation of␣v␤3also downmodulates uptake of apoptotic cells speciﬁcally,and ligation of two other integrins,␣6␤1and␣1␤2,have the same effect.This shows that uptake of apoptotic cells is regulated by events that induce signalling through integrin receptors irrespective of whether or not they are directly associated with uptake.This raises the question whether uptake of apoptotic cells via␣v␤3reciprocally inﬂu-ences functions of integrins responsible for cell adhesion and facilitate the emigration of macrophages from an inﬂamed focus as described by Bellingan et al.19MATERIALS AND METHODSReagents.Recombinant human tumor necrosis factor(rhTNF)-␣, rhTGF-␤,and recombinant rat interferon(IFN)-␥were obtained from Boehringer(Ingelheim,Germany),Sigma Chemical Co(Dorset,UK), and Bradsure Biologicals Ltd(Loughborough,UK),respectively. Recombinant rat interleukin(IL)-4was produced in-house as described previously20using a Chinese hamster ovary(CHO)cell line generously donated by Dr Neil Barclay(MRC Cellular Immunology Unit,Oxford, UK).The rat monoclonal antibody(MoAb),F11,against the integrin␤3From the Department of Medicine and Therapeutics,University ofAberdeen,Aberdeen,UK.Submitted May8,1998;accepted October13,1998.Supported by Grant No.ER254/1-1from the Deutsche Forschungsge-meimschaft(to L.-P.E.),Grant No.044988/2/95/2from the WellcomeTrust(to G.M.W.),and the National Kidney Research Fund.Address reprint requests to Lars-Peter Erwig,MD,University ofAberdeen,Department of Medicine and Therapeutics,Institute ofMedical Sciences,Foresterhill,Aberdeen AB252ZD,UK;e-mail:L.P.Erwig@.The publication costs of this article were defrayed in part by pagecharge payment.This article must therefore be hereby marked‘‘adver-tisement’’in accordance with18U.S.C.section1734solely to indicatethis fact.௠1999by The American Society of Hematology.0006-4971/99/9304-0010$3.00/01406Blood,Vol93,No4(February15),1999:pp1406-1412chain21was a gift from Prof Michael Horton(Bone and Mineral Centre, University College London Medical School,London,UK).The mouse antirat integrin antibody␣6␤1,CD18,CD116,anti-CD45,anti-CD25, anti-ED3,and mouse antihuman CD21were obtained from Serotec (Oxford,UK).The rabbit antihuman erythrocyte membrane antibody was obtained from DAKO(Glostrup,Denmark).The tetrapeptides arg-gly-asp-ser(RGDS),arg-gly-glu-ser(RGES),and phospho-L-serine were obtained from Sigma Chemical Co.Isolation and culture of BMDM.Rat BMDM were obtained using a technique previously described in detail.22Brieﬂy,bone marrow cells wereﬂushed aseptically from the dissected femurs of male Spraque Dawley rats with a jet of complete medium directed through a25-gauge needle to form a single cell suspension.The cells were cultured in Dulbecco’s modiﬁed Eagle’s medium(DMEM)containing2mmol/L glutamine,100U/mL penicillin,and100U/mL streptomycin,10% heat-inactivated fetal calf serum,and10%L929conditioned medium as a source of macrophage-colony stimulating factor(M-CSF).After7 days in culture,BMDM were dispensed into24-well culture plates (Corning,Corning,NY)at a concentration of5ϫ105cells/well and rested in medium without added M-CSF for24hours before use in experiments.Inhibition of uptake of apoptotic neutrophils.BMDM were incu-bated with a series of inhibitors at concentrations of1mmol/L for15 minutes at4°C and were washed immediately before interaction with apoptotic neutrophils.Phospho-L-serine was used as a stereo-speciﬁc inhibitor of the macrophage phosphatidylserine receptor,using condi-tions described by Fadok et al.5The tetrapeptide arg-gly-asp-ser (RGDS)was used as described,4and the noninhibitory peptide arg-gly-glu-ser(RGES)added as a control.Assay for uptake of apoptotic neutrophils.BMDM were transferred to24-well plates at a density of5ϫ105cells/well and rested for24 hours before the medium was changed and the cells incubated with various cytokines.Uptake of apoptotic neutrophils was assessed after 48hours using a microscopically quantiﬁed phagocytic assay,which has previously been described and illustrated in detail.4,23Apoptotic neutrophils were prepared from PMN isolated from fresh heparinized normal human blood by dextran sedimentation and Percoll centrifuga-tion.They were aged in teﬂon bags for approximately24hours in RPMI 1640supplemented with antibiotics and10%fetal calf serum.More than98%of these cells excluded trypan blue while apoptosis was veriﬁed by oil immersion light microscopy of May-Giemsa–stained cytospin preparations as previously described.23The apoptotic cells were washed once and resuspended in RPMI at a concentration of2.5ϫ106/mL.A total of1mL of cells was added to each well and allowed to interact with the macrophages for30minutes at37°C in a5%CO2 atmosphere.The wells were washed in saline at4°C to remove noningested PMN,ﬁxed with2%gluteraldehyde in0.9%saline,and stained for myeloperoxidase to identify ingested PMN.The proportion of macrophages that had ingested neutrophils was then counted by inverted light microscopy.To determine the effect of previous ingestion of apoptotic neutrophils on macrophages,rat BMDM were transferred to24-well plates at a density of5ϫ105cells/well and rested for24hours before the medium was changed and cells were incubated with either medium alone,RGDS peptide followed by apoptotic neutrophils,or apoptotic neutrophils alone.After30minutes incubation,the wells were washed in saline at 4°C to remove noningested PMN,and the macrophages were rested for 48hours in control medium or medium containing various cytokines. They were then reincubated for30minutes with a second challenge of apoptotic neutrophils and the proportion of macrophages that took up PMN assessed.The assay for uptake of opsonized erythrocytes was performed exactly as previously described.24Ligation of the integrin receptors.MoAbs to␣v␤3,␣6␤1,␣1␤2, CD25,and CD45were used to assess the effect of ligation of macrophage cell surface receptors on uptake of apoptotic neutrophils.Macrophages were incubated at various MoAb concentrations ranging from0.01to10µg/mL,for30minutes,at4°C in saline,or were incubated with various concentrations of mouse antihuman CD21as an irrelevant isotype-matched control.The cells were then washed before incubation in medium for various times before the start of the standard interaction assay with apoptotic PMN.Quantitation of nitric oxide(NO)generation.Generation of NO was measured by assaying culture supernatants for nitrite,a stable reaction product of NO.Aliquots of200µL of each cell-free culture supernatant were incubated with50µL of Griess reagent(0.5% sulphanilamide,0.05%N-(1-naphtyl)ethylendiamine dihydrochloride in2.5%phosphoric acid)in96-ﬂat–bottomed tissue culture plates for 10minutes at room temperature.The optical densities of the assay samples were then measured at540nm using a solution of phenol red free DMEM.In most experiments,nitrite was measured48hours after exposure to cytokines.RESULTSCytokines regulate uptake of apoptotic neutrophils by BMDM. The initial experiment was designed to conﬁrm our previous observations that pro and antiinﬂammatory cytokines inﬂuence uptake of apoptotic human neutrophils by uncommitted rat BMDM.20TNF caused a36%increase in the proportion of BMDM that took up apoptotic neutrophils compared with controls,whereas IL-4,IL-10,and IFN-␥inhibited uptake by 56%,22%,and42%,respectively,and TGF-␤had no effect (Table1).Incubation with cytokines modulated not only the number of macrophages taking up apoptotic cells,but also the average number of neutrophils per macrophage,ie,IL-4caused a56%decrease in the number of macrophages taking up apoptotic neutrophils and a40%reduction in the number of neutrophils per macrophage.Theseﬁndings differ from those reported for human monocyte-derived macrophages.In these cells,incubation with proinﬂammatory cytokines(IFN and TNF)increased their ability to ingest neutrophils,whereas antiinﬂammatory cytokines(IL-4,IL-6,and IL-10)had no effect.25These differences could reﬂect the source and species of the macrophages used or the conditions in which they were matured.Recently,Bonder et al26have shown that human 7-day–cultured monocytes did not express the functionally active IL-2receptor␥-chain,a component of the IL-4receptor, whereas macrophages did,which may explain the different effect of IL-4on uptake of apoptotic neutrophils by monocyte-derived macrophages and BMDM.BMDM use an integrin-dependent mechanism to recognize apoptotic PMN.Human monocyte-like cell lines and murine peritoneal macrophages use the phosphatidylserine receptor Table1.Effect of Cytokines on the Number of Macrophages ThatTake up Apoptotic NeutrophilsCytokine(Concentration)Uptake of Apoptotic PMNs(%)Control31Ϯ2.8IFNϩTNF(20U/mL,10ng/mL)18Ϯ1.9*IFN(20U/mL)21.6Ϯ2†TNF(10ng/mL)42.6Ϯ2.8*TGF-␤(7.5ng/mL)28.2Ϯ2.5IL-4(5µL/mL)13Ϯ2.5*IL-10(100ng/mL)22.8Ϯ2.7Nϭ10.*PϽ.01relative to unstimulated controls.†PϽ.05relative to unstimulated controls.MODULATION OF NEUTROPHIL UPTAKE BY MACROPHAGES1407(PSR)for recognition of apoptotic cells.27Human monocyte-derived macrophages and murine BMDM have been reported to use the␣v␤3/CD36/thrombospondin pathway.3In our studies,1 mmol/L RDGS speciﬁcally inhibited uptake of apoptotic PMN by unstimulated rat BMDM and by macrophages incubated for 48hours with IFN-␥,TNF,IL-4,or TGF-␤.Neither the control peptide RGES,nor phospho-L-serine,which inhibits PS-mediated recognition of apoptotic cells,5had any effect on uptake by cytokine-stimulated or unstimulated macrophages (Table2).Thus,both uncommitted or cytokine-stimulated rat BMDM use an integrin-dependent recognition mechanism, presumptively the CD36/␣v␤3/thrombospondin system rather than a PSR-dependent mechanism.This conclusion is strength-ened by the demonstration of␣v␤3on the surface of BMDM by immunoﬂuorescence using the MoAb,F11,directed against the ␤3subunit of the receptor(data not shown).To verify the recognition mechanism,it would be necessary to block either CD36or␣v␤3on the macrophage surface.To our knowledge, the one antirat antibody available for this purpose is the mouse MoAb F11against the␤3subunit of the vitronectin receptor, which is a poor blocking antibody under our experimental conditions.BMDM incubated with F11for45minutes and then seeded in vitronectin-coated plates adhered as efficiently as control macrophages.There was no difference in the number ofnonadherent cells(less than1%of the seeded cells in both groups)when aliquots of the supernatants of control and F11-treated macrophages were examined2,4,12,and24hours after seeding(data not shown).Previous uptake of apoptotic PMNs reduces the ability of BMDM to ingest apoptotic PMN.To determine the effect of uptake of apoptotic neutrophils on macrophage function,uncom-mitted rat BMDM were challenged for30minutes with apoptotic neutrophils in medium alone or in the presence of RGD peptide to prevent uptake.They were then rested for48 hours in medium before being reexposed to freshly prepared apoptotic neutrophils.Macrophages that had previously in-gested apoptotic PMN had a markedly reduced ability to engulf apoptotic neutrophils compared with control macrophages (Fig1),whereas their ability to take up opsonized erythrocytes was unchanged(data not shown).The difference cannot be attributed to a nonspeciﬁc effect of the neutrophils because macrophages challenged with PMN in the presence of RGDS-peptide retain their subsequent ability to take up apoptotic neutrophils(Fig1).The degree of inhibition was comparable to that observed when BMDM are exposed to IFN-␥,IL-4,or IL-10,which we have previously shown cannot be reversed by treatment with TNF.28By contrast,uptake of apoptotic cells by BMDM did not abrogate the modulatory effects of TNF or other cytokines on uptake of apoptotic cells when added to the medium after the initial challenge.However,in each,their capacity to take up apoptotic PMNs was reduced by50%.Furthermore,prior uptake of PMNs did not affect the ability of IFN-␥to prime macrophages for generation of NO(Table3).In this set of experiments,there was no signiﬁcant difference in IFN/TNF-induced NO generation between uncommitted BMDM,macro-phages that had ingested apoptotic neutrophils,and macro-phages that have been incubated with RGDS peptide followed by apoptotic neutrophils.Thus,uptake of apoptotic cells speciﬁcally inhibits BMDM ability to engulf apoptotic cells without interfering with their ability to respond to a range of pro and antiinﬂammatory cytokines.Ligation of the␣v␤3receptor and other integrins reduce uptake of apoptotic PMNs.Overloading the macrophage phagocytic capacity provides the most obvious explanation as to why uptake of apoptotic cells prevented further uptake.Table2.Effect of Inhibitors on the Number of Macrophages Takingup Apoptotic PMN(%)ControlRGDS(1mmol/L)RGES(1mmol/L)Phospho-LSerine(1mmol/L)Control32Ϯ2.49.2*Ϯ130.6Ϯ228.2Ϯ2.2 IFN-TNF(20U/mL,10ng/mL)18.0Ϯ2.37.9*Ϯ1.517.9Ϯ4.816.3Ϯ3.2 TNF(10ng/mL)42.6Ϯ2.415*Ϯ1.839.7Ϯ3.143Ϯ3 TGF(7.5ng/mL)28Ϯ4.214.1†Ϯ2.127.5Ϯ3.324.8Ϯ3IL-4(5µL/mL)12.6Ϯ2.2 5.7†Ϯ1.913.5Ϯ112.1Ϯ1.3This table shows the effect of inhibitors on recognition of apoptotic PMNs by control and cytokine-stimulated BMDM.*PϽ.01relative to controls.†PϽ.05relative tocontrols.Fig1.Figure1shows the percentage uptake of apoptotic neutro-phils by BMDM.The macrophages were incubated48hours before the interaction assay with apoptotic neutrophils,RGDS followed by apoptotic neutrophils or medium.They were then washed and cultured in medium containing cytokines or medium alone before washing and a30-minute interaction with apoptotic PMN;mean؎standard error(SE),n؍10;*P F.01.Table3.Effect of Uptake of Apoptotic PMN or Ligation of Integrins on IFN/TNF-Induced NO Generation(Arbitrary Units)PMNRGDS(1mmol/L)ϩPMN MediumF11(1µg/mL)CD18(1µg/mL) IFN-TNF(20U/mL,10ng/mL)21.2Ϯ1.223.4Ϯ0.922.7Ϯ1.119.4Ϯ3.422.5Ϯ1.9 Control 2.1Ϯ1.2 2.9Ϯ0.5 2.5Ϯ0.7 2.09Ϯ0.9 1.8Ϯ1.11408ERWIG ET ALHowever,this seems unlikely for three reasons.First,uptake of opsonized erythrocytes did not downmodulate the ability of macrophages to ingest apoptotic neutrophils 48hours later (Table 4).Second,incubation of BMDM with neutrophils from different donors known to induce high or low uptake showed that irrespective of whether 20%or 40%of macrophages took up apoptotic cells and regardless of substantial differences in the number of PMN ingested per macrophage,the degree of downmodulation was about 50%(data not shown).Finally,the 48hours between the PMN challenges should be sufficient to allow the macrophages to recover.The fact that inhibition was prevented by incubation in the presence of RGDS suggested that the mechanism might involve the ␣v ␤3/CD36/thrombospon-din pathway.4This was addressed by incubation of BMDM for 30minutes with various concentrations of the MoAb,F11,against the ␤3subunit of the ␣v ␤3receptor.21F11blocks the calcium response after peptide binding to the vitronectin receptor in rat osteoclast.29It binds to ␣v ␤3on the surface of macrophages,but did not alter their adhesion to vitronectin,nor did it block uptake of apoptotic PMN by BMDM.Despite this,ligation of ␣v ␤3with F1112to 36hours before the interactionassay caused substantial reduction of PMN uptake (Fig 2),comparable to that induced by apoptotic PMN themselves.These data indicate that ligation of ␣v ␤3integrin decreases uptake of apoptotic neutrophils after a delay of at least 90minutes,whereas an isotype-matched control mouse antihuman CD21MoAb,which recognized neither PMNs nor macro-phages,had no effect.To examine the speciﬁcity of the effects of F11,the experiments were repeated,ﬁrst using MoAb against ␣6␤1,another integrin receptor expressed by macrophages,but not known to be involved in recognition of apoptotic neutro-phils,and secondly using a MoAb against CD45,another molecule on the macrophage plasma membrane.Strikingly the MoAb against ␣6␤1also downregulated uptake of PMNs with identical kinetics to antibodies against ␣v ␤3,whereas the MoAb against CD45had no effect (Fig 3).Thus,ligation of integrins,but not of other receptors on the surface of macrophages,speciﬁcally downregu-lates uptake of apoptotic cells after a delay of more than 90minutes,but did not affect the uptake of other particles such as opsonized red blood cells (data not shown).To conﬁrm these observations,we performed another set of experiments examining the role of receptor ligation on uptake of apoptotic PMNs by macrophages after ligation of the receptors for 45minutes 12hours before the interaction assay.Ligation of the integrin receptors CD11b (percentage uptake 14.4Ϯ2.1,P Ͻ.01)and CD18(13.4Ϯ3.4,P Ͻ.01)signiﬁcantly decreased uptake of apoptotic cells by macro-phages,whereas incubation with MoAbs against the IL-2receptor CD25(28.7Ϯ3.5)present on the macrophage surface,or ED3(29Ϯ4),only expressed by activated or tissueTable 4.Effect of Uptake of Opsonized Erythrocytes on the Number of Macrophages That Take up Apoptotic Neutrophils 48Hours LaterInitial ChallengeUptake of Apoptotic PMNs (%)Control25.8Ϯ7.3Opsonized erythrocytes 27.2Ϯ4.8N ϭ5.Fig 2.Figure 2shows the effect of ligation of ␣v ␤3(1␮g/mL)and an isotype-matched control CD21(10␮g/mL)on uptake of apoptotic neutrophils by BMDM.The macrophages were incubated for 30minutes at various times before the start of the interaction assay.Mean (percentage of uptake)؎SE,n ؍8.*P F.01.Fig 3.Figure 3shows the effect of ligation of ␣6␤1and CD45(10␮g/mL)on uptake of apoptotic neutrophils by BMDM.The macro-phages were incubated for 30minutes at various times before the start of the interaction assay.Mean (percentage of uptake)؎SE,n ؍8.*P F .01.MODULATION OF NEUTROPHIL UPTAKE BY MACROPHAGES 1409macrophages,were not different from controls(29.8Ϯ3.4). Thus,ligation of three different integrin receptors speciﬁcally downmodulated macrophage ingestion of apoptotic neutrophils, whereas ligation of two other receptors on the macrophage surface did not.It seems likely that at least some of the modulating effects of apoptotic PMNs themselves can be attributed to this mechanism.In addition,there was no signiﬁ-cant difference in IFN/TNF-induced NO generation between uncommitted BMDM and macrophages,which have been incubated with antibodies that ligate their integrin receptors (Table3).Thus,similar to uptake of apoptotic cells,ligation of integrin receptors speciﬁcally inhibits BMDM ability to engulf apoptotic cells without interfering with their ability to respond to a range of pro and antiinﬂammatory cytokines.DISCUSSIONThe speciﬁc uptake of apoptotic neutrophils by macrophages is one of the critical steps in the resolution of inﬂammation.30It provides a way to remove neutrophils before granulocyte lysis and release of the neutrophils’cytotoxic contents16and does not activate the macrophages usual proinﬂammatory response to phagocytosis.Indeed,Fadok et al17have recently provided evidence that uptake of apoptotic cells induces macrophages to synthesize the antiinﬂammatory cytokine,TGF-␤.The impor-tance of the process is illustrated by the observation that insufficient or impaired capacity for phagocytic clearance leads to disintegration of the cells undergoing apoptosis and worsen-ing of tissue damage.31We hypothesized that alterations in the process responsible for removal of apoptotic neutrophils might contribute to these observations.In some situations,induction of a single episode of acute inﬂammation resolves quickly, whereas a second episode results in progressive tissue damage. One explanation for this might be that the difference was caused by a reduced capacity to remove apoptotic neutrophils.32This prompted us to analyze the effect of ingestion of apoptotic cells on the ability of macrophages to take up a second pulse of apoptotic cells48hours later.The results show that uptake of the second pulse48hours after theﬁrst is consistently reduced by50%,which could have a substantial effect on tissue repair. The characteristics of the mechanisms responsible for im-paired uptake demonstrate that it involves speciﬁc interaction between the neutrophils and macrophages:(1)neutrophil up-take by BMDM was inhibited by RGDS,which interrupts integrin-dependent recognition;(2)it was not inﬂuenced by uptake of opsonized erythrocytes and was independent of the magnitude of theﬁrst‘‘neutrophil meal’’and thus unlikely to be due simply to macrophage‘‘indigestion’’;(3)the effect was sustained for at least48hours;(4)it did not interfere with cytokine-induced modulation of uptake of apoptotic cells;and (5)uptake of apoptotic neutrophils does not inﬂuence IFN-␥/ TNF-induced generation of NO by macrophages.Taken to-gether,these characteristics suggest that modulation is caused by the speciﬁc interactions between macrophage receptors and ligands on the PMN.A number of different macrophage receptor-mediated path-ways have been described to be involved in uptake of apoptotic neutrophils:(1)an uncharacterized lectin-dependent interac-tion2;(2)a complicated charge sensitive process involving the CD36/vitronectin receptor(␣v␤3)complex on the macrophage surface interacting with unknown moieties on the apoptotic PMN surface via a thrombospondin bridge3,4;(3)a stereo-speciﬁc recognition of phosphatidylserine that is expressed on the surface of the apoptotic cell after loss of membrane asymmetry5,6;(4)macrophage scavenger receptors7;(5)the LPS receptor,CD148-10and macrosialin or CD68.11,12Inhibition by RGDS,but not PS,suggests that uptake by rat BMDM in our experiments is mediated by the␣v␤3/CD36/thrombospondin recognition pathway,which has been extensively characterized by Savill et al.3,4The importance of␣v␤3was identiﬁed in blocking experiments using MoAbs.Our experiments were conducted using the MoAb,F11,an antibody to the␤3chain, which blocks some␣v␤3-dependent functions,but not the ability of BMDM to bind to vitronectin under our experimental conditions.Despite this,it caused a sustained downmodulation of the macrophages’ability to take up apoptotic cells after a delay of more than90minutes,comparable in degree to that seen after ingestion of apoptotic cells.This effect was not observed with isotype-matched control antibodies or with antibodies to CD25or CD45on the macrophage surface. Strikingly,however,antibodies to three other integrin receptors,␣6␤1,CD11b,and CD18,not known to be associated with uptake of apoptotic neutrophils,had the same effect.Thus, ligation of␤1,␤2,and␤3integrins all cause sustained speciﬁc downmodulation of uptake of apoptotic cells,but no effect on uptake of opsonized erythrocytes,and presumptively␤1and␤2 integrins downmodulate the function of␣v␤3in neutrophil uptake.There are precedents for cross-inhibition between integrin receptors,including interactions involving␣v␤3.Blystone et al33have previously shown that ligation of␣v␤3blocks high-affinity phagocytic function,but not adhesive function of theﬁbronectin receptor␣5␤1,possibly by inﬂuencing serine/ threonine kinase activity of the cytoplasmic portion␤1chain. Similarly␣4␤1ligation inhibits␣5␤1-dependent expression of metalloproteinases.34Diaz-Gonzalez et al35and Fenczik et al36 have analyzed the cross-talk between different integrins in detail and introduced the term transdominant inhibition to describe this phenomenon.They showed that ligation of␣ll b␤3 suppresses adhesive properties of␣5␤1and␣2␤1.35,36They demonstrated that the phenomenon was dependent on the assumption of the high-affinity state of␣ll b␤3and was attribut-able to conformational changes in the cytoplasmatic portion of the␤1chain.37It is not yet clear at what level changes in integrins might modulate uptake of apoptotic cells,partly because of uncertainties about the signalling pathways in-volved.The intracellular signalling pathways that control uptake of apoptotic cells have not been studied systematically.However, Rossi et al38have recently reported that activation of cyclic adenosine monophosphate(cAMP)signalling pathways by inﬂammatory mediators downmodulates macrophage ingestion of apoptotic cells and that alteration in cAMP concentrations might be responsible for the observation that ligation of CD44 speciﬁcally enhances phagocytosis of apoptotic neutrophils.39 Our results suggest that uptake is also regulated by cross-talk between integrins and emphasize the multiple levels of control for macrophage removal of apoptotic cells.The ability of macrophages to ingest apoptotic cells can be dynamically1410ERWIG ET AL。

病例报告英语作文

病例报告英语作文Title: A Case Report: The Diagnosis and Treatment of a Rare Medical Condition。

Abstract:This case report presents a rare medical condition of a 45-year-old patient who presented with a unique set of symptoms. The aim of this report is to describe the diagnostic process, treatment plan, and patient outcome. The case highlights the importance of a multidisciplinary approach and thorough investigation in diagnosing and managing rare medical conditions.Introduction:Rare medical conditions pose significant challenges in terms of diagnosis and treatment due to their limited prevalence and diverse clinical presentations. This case report focuses on a patient with an unusual set of symptomsthat required a comprehensive evaluation to determine the underlying cause.Case Presentation:A 45-year-old male patient presented with a three-month history of fatigue, weight loss, and intermittent fevers. The patient reported no significant medical history or family history of similar symptoms. Initial physical examination revealed enlarged lymph nodes in the neck and groin, along with hepatomegaly. Laboratory investigations showed elevated inflammatory markers and abnormal liver function tests.Diagnostic Assessment:Given the patient's symptoms and physical examination findings, a wide range of potential diagnoses were considered, including infectious, autoimmune, and neoplastic diseases. The patient underwent a series of diagnostic tests, including blood cultures, serology for infectious diseases, imaging studies, and a lymph nodebiopsy.Results:Blood cultures were negative for any bacterial orfungal growth. Serology tests ruled out common infectious diseases such as tuberculosis and HIV. Imaging studies revealed multiple enlarged lymph nodes in various regionsof the body. A lymph node biopsy was performed, and histopathological examination showed features consistentwith Castleman disease, a rare lymphoproliferative disorder. Treatment and Outcome:The patient was referred to a multidisciplinary team consisting of hematologists, oncologists, and infectious disease specialists for further management. The treatment plan included the administration of corticosteroids to alleviate symptoms and reduce inflammation. Additionally,the patient received targeted therapy with rituximab, a monoclonal antibody, to target the abnormal lymphocytes.Over the course of several months, the patient showed significant improvement in symptoms, with a reduction in lymph node size and normalization of liver function tests. Regular follow-up visits were scheduled to monitor the patient's progress and adjust the treatment plan as necessary.Discussion:Castleman disease is a rare disorder characterized by abnormal lymph node enlargement and systemic symptoms. The diagnosis of this condition requires a combination of clinical suspicion, thorough investigation, and histopathological examination. Treatment options vary depending on the subtype of Castleman disease and may include surgery, chemotherapy, or targeted therapy.Conclusion:This case report highlights the importance of a multidisciplinary approach in diagnosing and managing rare medical conditions. The successful diagnosis and treatmentof Castleman disease in this patient demonstrate the significance of thorough investigation, collaboration among healthcare professionals, and individualized treatment plans. Further research is needed to enhance our understanding of rare medical conditions and improvepatient outcomes.。

华师细胞生物学名词解释(个人总结)

19、结合变构机制（binding change mechanism）:与ATP合酶活性密切相关的3个β亚基各自具有一定的构想，不同构象与核苷酸结合的亲和力也不同，在ATP合成过程中，在H+流驱动γ和ε亚基旋转120度的情况下，3个β亚基的构象也相互发生转变，从而引起β亚基对ATP、ADP和Pi亲和力的改变，导致了ATP的合成和释放。
24、核孔复合体（nuclear pore complex）:核被膜上由外环、内环、幅和中央栓等多种核孔蛋白构成的联系核质和细胞质的复杂隧道结构，对进出核的物质有控制作用。
25、受体（receptor）:细胞膜表面或细胞内部存在的一类蛋白质，其上有专门位点和信号分子进行结合，结合后可以将信号分子携带的信息传递到胞内，引起细胞发生一系列反应。
6、踏车行为(tread milling)：在一定条件下，微管或微丝正极发生聚合而延长，负极发生结局而减短的现象。
7、减数分裂(meiosis)：有性生殖个体形成生殖细胞的一种分裂形成的子细胞的染色体数目减半。
8、细胞分化(cell differentiation)：在个体的发育中，由一种相同的细胞类型经细胞分裂后逐渐在形态、结构和功能上形成稳定性差异，产生不同细胞类群的过程称之。
17、第二信使学说（second messenger theory）：胞外化学物质（第一信使）不能进入细胞，它作用于细胞表面受体，而导致产生胞内第二信使，从而激发一系列生化反应，最后产生一定的生理效应，第二信使的降解使其信号作用终止。
18、细胞外被（cell coat）:又称糖萼，用来指细胞膜外表面覆盖的一层黏多糖物质，是细胞膜的结构成分。
28、原癌基因（proto-oncogene）:正常细胞中的病毒癌基因的同源物称为细胞癌基因或原癌基因，如生长因子受体、转录激活蛋白和核调节蛋白等，其突变可导致细胞生长失控，诱发癌症。

免疫学英文名词解释一

免疫学英文名词解释（一）1. Immunity: “a condition of being able to resist a particular disease especially through preventing development of a pathogenic microorganism or by counteracting the effects of its products”2. Immunology: Immunology is the study of our protection from foreign macromolecules or invading organisms and our responses to them.3. Innate immunity: evolves with the germline and involves receptors, enzymes and cells that detect conserved aspects of microbes and parasites. It is the 1st line of defense. No specificity, no memory.4. Adaptive immunity is provided by T & B lymphocytes. It is the 2nd line of defense. It has two important characteristics: Immune response is highly specific for the antigen that triggered it. Exposure to antigen creates an immunologic “memory.”5. Primary lymphoid organs: Lymphoid organs include primary (bone marrow & thymus) and secondary lymphoid organs and tissues (lymphoid nodes、spleen、MALT). Primary lymphoid organs are the place where lymphocytes develop and mature. Lymphocytes includes B cell and T cell, respectively originating from bone marrow and thymus and mediates humoral and cellular immunity.6. Secondary lymphoid organs: Secondary lymphoid organs are the place that immune responses happen, which include lymph nodes, spleen, tonsil and MALT. Lymph nodes drain the connective tissues of the body. The spleen drains the blood. MALT are responsible for local infection.7. MALT: The majority (50%) of lymphoid tissue in the human body is located within the lining of the major tracts, including respiratory, digestive and genitourinary tracts. This is because these are the main sites of entry for microbes into the body. These are collectively called the mucosa-associated lymphoid tissues (MALT).8. Antigens are molecules which are recognized by receptors on lymphocytes. B lymphocytes usually recognize intact antigen molecules, while T lymphocytes recognize antigen fragments on the surface of antigen presenting cells.9. Epitope:Antigen molecules each have a set of antigenic determinants, also called epitopes. Epitopes are molecular shapes recognized by antibodies and T cells of the adaptive immune system.10. Clonal selection: Each lymphocyte is genetically programmed to be capable of recognizing essentially only one particular antigen. When an antigen binds to the cell that can recognize it, it is induced to proliferate rapidly. Within a few days there are a sufficient number to mount an adequate immune response. In another words, the antigen selects for and generates the specific clones of its own antigen-binding cells, a process called clone selection. In brief: clone selection involves recognition of antigen by a particular lymphocyte, this lead to clonal expansion and differentiation to effector and memory cells11. Opsonization: This s is a process of making a microbe easier to phagocyte. A number of molecules called “opsonins” do this by coating the microbes and aid attachment of the microbe to the phagocyte and also trigger activation of phagocytosis. Opsonins include the complement component C3b and antibody which acting as a bridge between antigen and phagocytes.12. NK cells: belong to lymphocyte family. But in contrast to all T and B lymphocytes, NK cell do not express antigen-specific receptors and do not possess the adaptive property of memory cell development: they are there for considered to form part of the innate immune system. However, like Tc, their main function is to kill infected cell and tumor cells using similar mechanism to those of Tc cells to induce apoptosis of their targets. NK cell are also able to kill targets coated with IgG via their receptor for IgG. This property is referred to as ADCC.13. DCs are required by T cell to enable them to respond to antigens. DCs are most important antigen presenting cells known so far and are the interface of innate and adoptive immunity. Functions: Antigen up-taking in peripheral sites & antigen presentation in lymph nodes.14. Complement: The complement system is an important component of innate immunity It can be activated by the classical and alternative pathways, both pathways will eventually lead to the lytic pathway which featured by the formation of MAC.Function of complement: anaphylaxis (C3a,C5a), chemotaxis (C5a)，opsonization (C3b,C4b), lysis (C56789)15. Interferons: Interferons are proteins involved in protection against viral infections. The two kinds of interferon, type I and type II, have different cellular origins and mediate a range of different activities. They interfere with viral replication but also are signaling molecules between cells.16. lymphocyte traffic and recirculation Lymphocytes produced in the primary lymphoid organs (thymus-T, bone marrow-B) migrate via the bloodstream to the secondary lymphoid organs/tissues where they carry out the function. They do not stay in one site but continually recirculate through the body in search of antigens.17. Affinity is the tightness of binding of an antibody binding site to an antigenic determinant (epitope)----the tighter the binding, the less likely the antibody is to dissociate from antigen.18. Valence: Valence is the maximum number of epitopes with which the antibody can react.19. Avidity: antibody binds a multivalent antigen is termed avidity, to differentiate it from the affinity of a single antigenic determinant for an individual combining site. Antibody avidity indicates the overall strength of interaction between antibody and antigen.20. Isotype: These are genetic markers on immunoglobulins shared by all the individual of same species. The genes for isotypic variants are present in all healthy members of a species. For example, the genes for γ1, γ2,γ3, γ4, μ, α1, α2, δ, ε, κ and λ chains are all present in the human genome, and are therefore isotypes.21. Allotype:These are genetic markers on immunoglobulins that segregate within the species. This refers to genetic variation between individuals within a species. For example, the variant of IgG3 called G3m(b0) is characterized by a phenylalanine at position 436 of the γ3 heavy chain. It is not found in all people and is therefore an allotype. Allotypes occur mostly as variants of heavy chain constant regions.22. Idiotype: These are unique antigenic determinant associated with antigen binding sites of antibodies and are the results of the different amino acid sequences which determine their specificities. Variation in the variable domain, particularly the hypervariable regions, produces idiotypes. These determine the binding specificity of the antigen-binding site.23. CDRs: At the amino acid level, the variable region of antibody is comprised of three regions of extreme variability (hyervarible region). They are called complementarity-determining regions, or CDRs.24. FRs: Interspersed among the CDRs are framework regions (FRs) which are less variable and more evolutionarily conserved. At the three-dimensional level, the three CDRs of each chain converge to form a combining site which recognize the antigenic determinant (epitope).25. Monoclonal antibody: In 1975, Kohler and Milstein developed a procedure to create cell lines producing predetermined, monospecific and monoclonal antibodies. The basic technology involves fusion of an immortal cell (a myeloma tumor cell) with a specific predetermined antibody-producing B cell from immunized animals or humans. The resulting hybridoma cell is immortal and synthesizes homogeneous, specific, mAb which can be made in large quantities.26. ADCC：ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY –The linking of antibody bound to target cells (virus infected cells, or some tumor cells) with FcR of natural killer cells (NK cells), neutrophils, macrophages,or eosinophils can result in killing of the target cell.27. cytokines: Definition: Cytokines are small molecules, secreted by cells in response to a stimulus. Function: As a group, cytokines induce growth, differentiation, chemotaxis, activation and/or enhanced cytotoxity. They are used for strengthening communications between cells.28. Toll like receptors (TLR)Toll like receptors are a family of proteins of which there are at least 5 known members.Using TLRs, innate immune cells can detect and respond to infection by recognizing conserved motifs of microbes. TLRs transmit signals about microbial constituents to the nucleus, thus regulating the type of genes expressed, and the subsequent response.29. Positive selection of T cells:T cells that express a TCR that can bind weakly to self MHC are spared from death and are positively selected to survive.30. Negative selection of T cells: T cells that react strongly to self-antigens on MHC are eliminated. Only those T cells that can react to MHC, but do not bind strongly to self-antigens emerge as mature T cells from the thymus.。

白细胞分化抗原及其单克隆抗体的临床应用(一)

白细胞分化抗原及其单克隆抗体的临床应用(一)白细胞分化抗原及其单克隆抗体的临床应用白细胞分化抗原的概述•白细胞分化抗原（Leukocyte Differentiation Antigen，简称LDA）是人体内参与免疫反应的一类表面分子。

•LDA的种类十分丰富，通常可以根据其分子结构和细胞类型进行分类。

•LDA的功能包括介导细胞间的相互作用、参与信号传递及分化调控等。

单克隆抗体的概述•单克隆抗体（Monoclonal Antibody，简称mAb）是一种具有高度特异性和亲和力的抗体。

•mAb通常由单一细胞克隆产生，因此具有单一的“特异性”。

•mAb的应用范围广泛，主要用于诊断、治疗和科研等领域。

LDA单克隆抗体在临床应用中的意义•LDA单克隆抗体具有高度特异性和亲和力，能够针对特定的LDA 亚型进行识别和标记。

•LDA单克隆抗体可以用于诊断和治疗多种疾病，如白血病、淋巴瘤、乳腺癌等。

•LDA单克隆抗体还可以用于科研领域，如信号通路研究、分化调控研究等。

LDA单克隆抗体的应用案例•CD20单克隆抗体：作为治疗NHL（非霍奇金淋巴瘤）的重要药物，已被广泛应用于临床。

•CD33单克隆抗体：用于治疗急性髓系白血病的药物，能够选择性杀死白血病细胞。

•CD52单克隆抗体：用于治疗多发性硬化症的药物，能够抑制免疫系统的异常反应。

总结•LDA单克隆抗体具有广泛的应用前景，是一类重要的生物制剂。

•随着医学技术的不断发展，相信LDA单克隆抗体在诊疗和科研领域的地位将越来越重要。

LDA单克隆抗体的研究进展•随着技术的不断进步，LDA单克隆抗体的研究也得到了更多的关注和深入探究。

•微纳米技术、基因工程技术等的应用，在LDA单克隆抗体的开发和制备方面起到了积极的作用。

•融合抗体技术、特异性免疫治疗等的发展，也为LDA单克隆抗体在疾病治疗中的应用提供了更加高效和准确的手段。

LDA单克隆抗体的未来展望•在临床应用方面，随着LDA单克隆抗体的不断发展和创新，更多种类和更好的质量可以被制备出来，使其在治疗和疾病诊断中的应用更加广泛和深入。

Ki-67抗原在人脑星形细胞肿瘤中的表达及其对预后的意义

Ki-67抗原在人脑星形细胞肿瘤中的表达及其对预后的意义丁涟沭;刘道坤;邓传宗;顾志恺;陈长;蔡用武;陈健;高宜录【期刊名称】《南通大学学报（医学版）》【年(卷),期】2001(021)001【摘要】Objective:To explore the prognostic factors for patients with astroc ytic tumors and to determine the value of Ki-67 labeling index obtained using MI B-1 monoclonal antibody in predictingsurvival.Methods:Data of 89 patients with astrocytic tumors were collected en rolled in Nantoing Medical College affiliated hospital from 1994 to 1996.Ki-67 w as determined with immunohistochemistry using monoclonal antibody MIB-1.For the univariate analysis,survival probabilities were estimated based on Kaplan-Meier 's Product-Limit Survival Estimates method and logrank test was used to assess THe association between patient survival and each variable.Multivariate regressio n analysis using Cox's proportional-hazards model was used to ascertain a simult aneous effect of outcome-related variables on survival.Results:the mean Ki-67 LIs were 2.67%±1.62% in GradeⅡ,6.74%±3.89% in GradeⅢ and 8.07%±3.84% in GradeⅣ.Analysis of variance indicated a signifi cant difference among them.Univariate analysis showed thaTHistologic grade and Ki-67 LI were significant factors for survival.Conclusion:There is a strong positive relationship between Ki-67 LI and histologic grade.The higher histologic grade,the higher Ki-67 LI.And Ki-67 L I>2.5% is a significant factor forshorter survival.In the same histologic grade ,there is a significant difference in patients' prognosis due to the different K i-67 LI.Whereas,with the different Ki-67 LI,some patients with differenTHistolo gic grade haveno significant survival time.Ki-67 LI and histologic grade detect ed together will provide more accurate useful information for determining the pa tients' prognosis.%目的：探讨Ki-67抗原在人脑星形细胞肿瘤中的表达及其对预后的意义。

东南大学农学院2021级《细胞生物学》考试试卷(4225)

东南大学农学院2021级《细胞生物学》课程试卷（含答案）__________学年第___学期考试类型：（闭卷）考试考试时间：90 分钟年级专业_____________学号_____________ 姓名_____________1、判断题（35分，每题5分）1. 叶绿体中，类囊体膜两侧也存在较高的电位差。

（）答案：错误解析：叶绿体中，类囊体膜不能使质子自由通过，所以导致膜两侧的浓度酸度差别较大。

2. 在原核细胞中，由DNA转录mRNA和由mRNA翻译蛋白质是同时并几乎在同一部位进行。

（）答案：正确解析：真核与原核细胞的一个非常明显显著差是：真核细胞核内转录，细胞质内翻译，具有严格的阶段性与区域化，而原核的转录与翻译同时、同地进行。

3. 激素受体都具有酪氨酸受体结构域。

（）答案：错误解析：激素受体都具有各自激素的特异结构域。

4. 所有的受体都是跨膜蛋白质。

（）答案：错误解析：表面细胞表面的糖蛋白多为跨膜糖蛋白，但细胞内的蛋白质受体如基因调控蛋白等不一定是。

5. 原核生物和真核生物细胞质膜内都含有胆固醇。

（）答案：错误解析：原核生物和植物线粒体的质膜没有胆固醇，只有动物细胞质膜中有。

6. 抑癌基因突变能转变成癌基因从而致癌。

（）答案：错误解析：抑癌基因发生突变后会失去抑癌功能。

7. 细胞外被是指与细胞膜中的蛋白质或脂类分子共价结合的糖链。

（）答案：正确解析：细胞外被又称糖萼，它是细胞膜的正常高负荷结构反应物，不仅对膜蛋白起为保护作用，而且在细胞识别中起重要作用。

2、名词解释（40分，每题5分）1. 胞质分裂答案：胞质分裂是指由在细胞分裂后半期末期时，通常于核分裂之后接着酿成的胞质体的一般来说分裂。

根据生物种类的不同，分崩离析胞质分裂可分为两个基本类型：一是在高等植物细胞中所，于细胞分裂的晚期，脉动姐妹染色单体群移到两极之后，纺锤体的中间区域分化为膜体。

在末期，从纺锤体中部形成细胞板；二是在动物细胞中，于突变的末期，赤道板位置上的表层细胞质部位向中间凹陷缢缩。

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Sensors2008,8, 582-593sensorsISSN 1424-8220© 2008 by MDPI/sensors Full Research PaperUsing Monoclonal Antibody to Determine Lead Ions with a Localized Surface Plasmon Resonance Fiber-optic BiosensorTsao-Jen Lin * and Mon-Fu ChungDepartment of Chemical Engineering, National Chung-Cheng University, Chia-Yi 621, Taiwan* Author to whom correspondence should be addressed. E-mail: chmtjl@.tw;Tel: 886-5-272044 ext 33405Received: 4 October 2007 / Accepted: 24 January 2008 / Published: 25 January 2008Abstract: A novel reflection-based localized surface plasmon resonance (LSPR) fiber-optic probe has been developed to determine the heavy metal lead ion concentration.Monoclonal antibody as the detecting probe containing massive amino groups to capturePb(II)-chelate complexes was immobilized onto gold nanoparticle-modified optical fiber(NMAuOF). The optimal immobilizing conditions of monoclonal antibody on to theNMAuOF are 189 µg/mL in pH7.4 PBS for 2 h at 25°C. The absorbability of thefunctionalized NMAuOF sensor increases to 12.2 % upon changing the Pb(II)-EDTA levelfrom 10 to 100 ppb with a detection limit of 0.27 ppb. The sensor retains 92.7 % of itsoriginal activity and gives reproducible results after storage in 5% D-(+)-Trehalosedehydrate solution at 4°C for 35 days. In conclusion, the monoclonal antibody-functionalized NMAuOF sensor shows a promising result for determining theconcentration of Pb(II) with high sensitivity.Keywords: localized surface plasmon resonance, biosensor, gold nanoparticle, monoclonalantibody, lead-chelate complex.1. IntroductionLead is one of the most widely used heavy metals and has a large number of industrial applications, including battery manufacture, paint, gasoline, alloys, radiation shielding, tank lining and piping. Among these, lead in paint and gasoline together accounts for most of the lead now in the environment and leads to serious pollution and human health problems. Elevated levels of lead can be harmful iflevels are built up in the body. Over the past several decades, it is found that lead is toxic by ingestion and inhalation, can affect the gut and central nervous system, and can cause anemia [1,2]. Besides, lead overexposure can also cause birth defects, mental retardation, behavioral disorders, and death in fetuses and young children [3,4]. Hence, detection and quantization of lead contamination is an important issue with the increasing requirement of environmental concern and health safety.Traditionally, the commonly used methods for determination of lead are atomic absorption (AA) spectroscopy [5] and inductively coupled plasma (ICP) optical emission spectrometry, and inductively coupled plasma mass spectrometry (ICPMS) [6,7]. These methods are sensitive and allow discrimination among different metal ions but require a tedious sample pretreatment and sophisticated instrument. Therefore, it is essential to develop a rapid and sensitive screening method to ascertain the presence of lead for in situ environmental monitoring. In the past, a lot of efforts have been devoted to develop rather inexpensive biosensors by using enzyme, peptide, protein, or antibody as recognition elements for the detection of heavy metals [8-12]. Theses recognition elements may be immobilized on a suitable surface or supporter, and its binding or inhibition can be measured by different detection techniques such as ion-sensitive field effect transistors, potentiometric and piezoelectric devices, amperometric electrodes, and optical-based biosensors. Among the recognition elements, monoclonal antibody provides a unique opportunity for distinguishing different metals and a basis for rapid and sensitive immunoassays [13-15]. To increase association with monoclonal antibody, small metal ions were initially conjugated with chelators to form a metal-chelate complex. The metal-chelate complexes represent a distinct class of hapten ligands which was recognized by specific amino acid residues in the antibody’s binding-sites to comprise a special subset of metal-binding protein [16,17]. Therefore, the specific binding characteristics of monoclonal antibody will be employed to our new design of biosensor for lead detection.A novel fiber-based biosensor with extraordinary optic properties of gold nanoparticles (NM Au) has recently been explored [18,19]. The absorption band of gold nanoparticles is obtained when the incident photon frequency is resonant with the collective oscillation of the conduction electrons and is known as the localized surface plasmon resonance (LSPR). The resonance frequency of the LSPR is highly dependent upon the local environment of the nanoparticles [20-22]. Therefore, the optical properties (e.g., absorbance and peak wavelength) of gold nanoparticles are sensitive to the refractive index of the surrounding solvent and the binding events to those functionalized nanoparticles. With a suitable receptor immobilized at the surface of the gold nanoparticles, the resulting LSPR fiber-optic sensor can detect the corresponding analyte even if the analyte is spectroscopically silent in the UV-vis region. Hence, the presence of analytes can be determined directly without the use of labeled molecules.For this study, a monoclonal antibody-functionalized fiber-based biosensor utilizing the LSPR effect was developed to evaluate the concentration of Pb(II). By using a self-assembling technique, a bioactive layer consisting of the monoclonal antibody was immobilized by covalent coupling onto the gold nanoparticle layer and the optimal conditions of immobilization will be examined. Prior to detecting, Pb(II) was treated with excess chelators (i.e., EDTA) to form Pb(II)-chelate complexes. The monoclonal antibody can exclusively bind with Pb(II)-chelate complex and causes a change in light attenuation. Based on the correlation between binding rate and light attenuation, the concentration ofPb(II) can be determined. Several factors, including stability and binding constant, were investigated as well.2. Materials and Methods2.1.Reagents and MaterialsMonoclonal Antibody to lead (K55061M) was purchased from Biodesign International Company, USA. Basically, Pb(II)-EDTA was covalently conjugated to keyhole limpet hemocyanin (KLH) and was used as an immunogen. Then, the KLH-Pb(II) conjugate was injected into mouse to produce the monoclonal antibody. Multimode plastic-clad silica optical fiber (model F-MBC) was purchased from Newport (Irvine, CA) with core and cladding diameters of 400 and 430 µm, respectively. The following chemicals, n-hexadecyltrimethylammonium bromide (CTAB, Fluka), sodium borohydride (Lancaster), 3-(mercaptopropyl)-trimethoxysilane (MPTMS, Acros), cystamine dihydrochloride (Sigma), phosphate buffered saline (PBS, Sigma), N-(3-Dimethylaminopropy)-N-ethylcarbodiimide hydrochloride (EDC, Sigma), N-hydroxy- succinimide (NHS, Sigma), ethylenediamine tetraacetic acid (EDTA, Sigma), lead(II) nitrate (Osaka, 99+%), D-(+)-Trehalose dehydrate (Sigma), were used as received. All aqueous solutions were prepared with water purified using a YMDI-100 water purification unit (Yeameei Membrane) with a specific resistance of 18 MΩ cm.2.2 Preparation of gold nanoparticle-modified optical fiberFirst, colloidal Au solution was prepared by mixing hydrogen tetracholoraurate (1.78 mL, 25.4 nM), 8.22 mL of chloroform, and 0.4 mL of a 0.02 M ethanol solution of CTAB for 10 min to form a 4.52×10-4 M hydrogen tetrachloroaurate solution. Freshly prepared NaBH4 ethanol solution (0.8 mL, 0.15 M) was added to the hydrogen tetrachloroaurate solution with vigorous stirring for 30 min. Then, the unclad portion (5 cm) of the optical fibers was cleaned for 30 min in a bath consisting of 3 volumes of 30% H2O2 and 7 volumes of concentrated H2SO4. The clean unclad portions of the optical fibers were then submerged into vials of 1% solution of MPTMS in toluene. After 8 h, the optical fibers were rinsed with methanol to remove unbound monomers from the surface. After thorough rinsing, the unclad portions of the optical fibers were immersed in Au solution for 5 h to form a self-assembled gold nanoparticle monolayer (NM Au) on the core surface. Subsequently, the modified optical fibers were rinsed sequentially with water, methanol, and chloroform. Figs. 1(a) and 1(b) were shown the 2-D and 3-D AFM images of surfaces on the prepared MPTMS and gold nanoparticle-modified optical fiber, respectively. From the images of Fig. 1(a), the surface modified by MPTMS became smoother with a 2.05 nm roughness. A Joel transmission electron microscopic (TEM) 1200EX was applied to observe the samples, which had been dispersed and allowed to dry on copper grids. Histograms derived from TEM image analysis showed that the mean diameter of Au nanoparticles was 9.6±2.3 nm, as shown in Fig. 1(b).(b)Figure 1. (a) Images of 2-D and 3-D AFM surfaces on the prepared MPTMS; (b) Electronmicrograph and particle size distribution of pure Au nanoparticles.2.3 Immobilization of antibody onto NM AuNM Au was initially modified to form a self-assembled monolayer (SAM) of cystamine by immersing NM Au in 0.02 M cystamine dihydrochloride (pH 7.4 in PBS) for 2 h to form an amine functional group. The cystamine-modified NM Au was further immersed in a PBS solution with containing 450 mM EDC, 90 mM NHS, and various concentrations of antibody for 2 h at room temperature, rinsed with PBS, and air-dried at room temperature. Then, the amine group of cystamine modified on the NM Au can couple with the activated succinimide esters reacting from antibody and NHS, and finally form an amino bond between cystamine and antibody, as shown in Fig. 2. The concentration of antibody, pH value of solution, and incubation time were varied to find the optimal immobilization conditions.CystamineEDC+NHSmodified opticalfiberfor detectingPb2+＋monoclonalantibody2h222222H2H2H2H22H222－C－O－C－－C－OHH2H2－C－O－C－－C－Ogold nanoparticleoptical fiber1% MPTMS in toluene8 h H2O2OFigure 2. The chemical reactions scheme carried out for covalent binding of monoclonalantibody to the fiber-based LSPR sensor.2.4 Pb(II) and other metals detection by antibody modified sensorsIn LSPR sensors, the light attenuation will be affected as the analyte molecules are captured or reacted by a molecular recognition element immobilized on the sensor surface.When Pb(II) was given with an excess of EDTA, all Pb ions were bound in a 1:1 Pb(II)-EDTA complex. It is because of the high binding affinity of EDTA to insure that all the Pb(II) in the sample form a complex with EDTA. Besides, the antibody exhibited little or no ability to bind to the metal-free EDTA. In this study, the reaction was carried out in standard solution as mentioned in Eq. (1). When Pb(II)-EDTA complex binds with antibody, the decreasing light intensity is correlated to the concentration of Pb(II). The degree of absorbability can be calculated according to the following formula:100(%)ityAbsorbabil10×−=III(1)where I0 and I1 are the averaged light intensity measured at the initial antibody activity without and with Pb(II)-EDTA complex, respectively. According to World Health Organization (WHO), the Pb(II) concentration in drinking-water should be less than 50 ppb. In this study, the detecting range of Pb(II) is chosen from 10 to 100 ppb in pH 7.4 PBS buffer solution. For comparison, other metals, including Cu(II), Ni(II), and Mg(II), had performed the same treatments and testing as Pb(II).2.5 Stability of antibody modified sensorsFor evaluate the stability, the immobilized LSPR sensors were immersed in a 5% D-(+)-Trehalose dehydrate solution and were stored in a 4°C refrigerator. The preserved sensors were taken out for thechange of calibration curve on Pb(II)-EDTA complex detection after 1, 7, 14, 21, 28, and 35 days. The relative activity for each time of test can be calculated by the following formula: 100(%)activity relative ×−=on o A A A (2) Where A o and A n were the change absorbabilities of the sensor on the initial manufactured day and the preserved n days, respectively.2.6 Instrumentation and measurementsThe optical configuration for the gold nanoparticle-modified optical fiber biosensor is a reflection-based fiber-optic configuration, as shown in Fig. 3. The system consists of a laser (Hitachi HL6320G laser diode, 635 nm, 10 mW; Thorlabs LDC500 laser diode controller; Thorlabs TEC2000 temperature controller; Thorlabs TCLDM9 laser mount), a chopper (Stanford Research SR540), a fiber coupler, a beam splitter, a sensing fiber, a liquid cell (10 mL), a photo-receiver (Thorlabs PDA55), and a lock-in amplifier (Stanford Research SR830). For the detail description of the biosensor system, please refer to Chau et al. (2006) [19]. The temperature of the liquid testing cell was maintained by a circulatory waterbath (Wisdom, model LC-06) within ±0.05 K. The Pb(II)-EDTA complex solution was filled in the liquid testing cell, in which the LSPR sensor was installed horizontally. After reaching a balance, a new stable response can be attained in 5 min. However, to make sure reaction reaching to equilibrium state, the responding signals are taken over 10 minutes after addition of the Pb(II)-EDTA complex solution. In this work, at least three replicates were performed for each parameter.A : Computer Lock-in amplifier Fiber coupler & CellPhoto receiver FiberLaser Temperature controlF : Beam splitterB : G :C : H :D : I : DA Figure 3. The schematic representation of the LSPR biosensor system.3. Results and Discussion3.1 Comparing probe w/wt immobilizing monoclonal antibody on NM AuWhile applying the LSPR sensor, the capture of Pb(II)-EDTA complex by the immobilized antibody causes a decrease of light intensity at the detector due to an increase in the local RI. Fig. 4 shows an example of serial responding signals from the antibody modified LSPR sensor immersing in a pH 7.4 PBS buffer solution after sequential addition of Pb(II)-EDTA complex to final concentrationsof 100 ppb. The responding signals decrease with increasing Pb(II)-EDTA complex concentration at room temperature and with a good linearity (which will be shown later). Notably, under the same operating conditions, there is no significant responding change for the fiber without immobilizing antibody, suggesting that the antibody can substantially differentiate the change of Pb(II)-EDTA complex at low concentrations.Figure 4. An example of serial Pb(II)-EDTA complex response signal in the range of10~100 ppb by two sensors with/without monoclonal antibody coating.3.2 Optimal monoclonal antibody immobilizing conditionsThe sensitivity of an optic-fiber sensor is related to the immobilizing conditions, such as the amount of immobilized ligand and pH value of the solution [23]. The amount of immobilized ligand is determined by the ligand concentration and incubation period. Fig. 5 shows the effects of the antibody concentration, and incubation time and pH on the response signals for Pb(II)-EDTA complex, respectively. In theory, higher incubating antibody concentration (or the longer the incubating time) revealed larger immobilization of monoclonal antibody onto the fiber, resulting in a stronger LSPR effect and a smaller I0. Besides, under a constant concentration of Pb(II)-EDTA complex the sensor immobilized with more antibody has the higher residua to react Pb(II)-EDTA complex with a smaller I1, results in a higher absorbability and resolution with a rather good linearity. From Fig. 5(a) and (b), it can be seen that as the incubating antibody concentration and time increases from 94.5µg/mL and 1 h, respectively, to 189 µg/mL and 2 h, the absorbability reaches a maximal value of 12.2 % with a good linearity (R2=0.994) for Pb(II)-EDTA complex concentration ranging from 10 to 100 ppb. However, upon further increase in the incubating enzyme concentration or time, the amount of immobilized antibody onto the fiber becomes over saturated and causes steric hindrance from binding with Pb(II)-EDTA complex, leading to a less response in absorbability.(a)(b)(c)Figure 5. Effects of (a) the incubating monoclonal antibody concentration, (b) incubatingtime, and (c) pH on the response for detecting 10~100 ppb Pb(II)-EDTA complex in pH7.4 PBS buffer solution at 25°C.Figure 5(c) shows the plots of the absorbability versus Pb(II)-EDTA complex concentration with different sensors incubated in various pH values of 189 µg/mL antibody solution for 2 h. In the lower pH, the coupling between succinimide esters formed by antibody and NHS and amine group oncystamine is less effective to form the amino covalent binding [24]. Hence, only the base conditions of incubation will be considered in the study. From Fig. 5(c), it shows that the maximal absorbability change is found at pH 7.4, after which the absorbability decreases again. This result can be attributed to two reasons: first, the protein denaturation of antibody in the higher pH condition may lead to its lower activity. Second, the charge of antibody becomes negative in the higher pH condition, which leads to over saturated immobilizing antibody onto the fiber. In summary, the optimal immobilization conditions for modifying the LSPR sensor are 189 µg/mL antibody and pH 7.4 for 2 h incubation, and will be applied for the rest of the studies. With the limit of detection (LOD) defined as the intensity at a Pb(II)-EDTA complex concentration that yields a signal-to-noise (S/N) ratio of 3, the LOD of the sensor with optimal immobilization is 0.27 ppb.3.3 Temperature effect and comparison with other metal ionsMonoclonal antibody is a protein, of which activity depends on temperature. The antibody’s binding and dissociation capabilities with metal-chelate complex are strongly affected by temperature. Fig. 6 shows the response of absorbability for detecting 10~100 ppb Pb(II)-EDTA complex under different detecting temperatures. The optimal detecting temperature for antibody responding to Pb(II)-EDTA complex is at 25°C, after that the change of absorbability becomes less. An increasing temperature may enhance both the binding and dissociation rate. However, based on above results, it can be inferred that the temperature effects on the dissociation rate between antibody and Pb(II)-EDTA complex is larger than on the binding rate.Figure 6. Effect of the detection temperature on the response for detecting 10~100 ppb Pb(II)-EDTA complex in pH 7.4 PBS buffer solution at 25°C.Another important issue on whether the monoclonal antibody-functionalized LSPR sensor is able to bind other metals needs to be addressed. Except Pb(II)-EDTA complex, the bindings of Mg(II)-, Cu(II)-, and Ni (II)-EDTA complexes to the sensor were also examined, as shown in Fig. 7. Basically, the sensor shows a rather good linearity for the above metal ions. However, for 10~100 ppb detectingrange the absorbability of these three metal-EDTA complexes was only 3% change, which was smaller than that of Pb(II)-EDTA complex (12.2%). That means the monoclonal antibody has a higher affinity to Pb(II)-EDTA complex.Figure 7. Comparison of the responses between different metal-chelate complexes (i.e.,Mg(II)-, Cu(II)-, and Ni(II)-EDTA) through the monoclonal antibody-functionalized LSPRbiosensor.3.4 Stability and dissociation constantNote that protein can cause structural folding and becomes denatured if the surface water on it is not conserved after a long-time storage. D-(+)-Trehalose dehydrate, known as mycose, is an alpha-linked sugar that has the merits of rehydration and anti-oxidation to perverse the activity of protein [25]. To prove the stability of our sensor, we performed the measurement of Pb(II)-EDTA complex using the sensors stored in 5% D-(+)-Trehalose dehydrate solution at 4°C for up to 35 days after the initial measurement. The result shows that the relative activity of the sensor only decayed 7.2% after being stored for 35 days. Although there is some loss of the sensor activity after each cycle of use, the sensor can be recalibrated to determine Pb(II)-EDTA complex concentration. There was no significant change in the calibration curve after 35-day storage.4. ConclusionA monoclonal antibody-functionalized LSPR biosensor bas been successfully developed for determining the concentration of Pb(II). The optimal incubating conditions for the immobilization of monoclonal antibody on to the LSPR biosensor were found to be 189 µg/mL monoclonal antibody in pH 7.4 PBS for 2 h. With the optimal modification, the sensor can obtain a maximal 12.2 % change of the absorbability for detecting 10~100 ppb Pb(II)-EDTA complex and a LOD of 0.27 ppb. The biosensor demonstrates a reproducible and stable response after over 35-day storage. 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