Study on Expression of HERG Protein in A549 Cell and Their Clinical Significance
Study_on_the_pharmacological_activities_and_chemic
ReviewStudy on the pharmacological activities and chemicalstructures of Viburnum dilatatumZhiheng Gao, Yufei Xi, Man Wang, Xiaoxiao Huang*, Shaojiang Song*Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research &Development, Liaoning Province, School of Traditional Chinese Materia Medica, ShenyangPharmaceutical University, Shenyang 110016, ChinaAbstractViburnum dilatatum (jiami in Chinese), belonging to the Caprifollaceae family, is widely distributed in Japan and China. Phytochemical investigations of Viburnum dilatatum (V. dilatatum) have resulted in the isolation of triterpenoids, phenolic glycosides essential oil, norisoprenoids, etc. Research results have shown that the chemical constituents of V. dilatatum possess various pharmacological activities, including antihyperglycemic, antioxidant activity and antiulcer effects. This study reviewed the chemical constituents and pharmacological activities of V. dilatatum to provide practical and useful information for further research and development of this plant.Keywords: Viburnum dilatatum; pharmacological activity; chemical structures1 IntroductionViburnum dilatatum (called jiami in Chinese, gamazumi in Japanese and snowball tree in English), beloinging to family Caprifoliaceae, is a deciduous low tree distributed widely in the hills of northern China and Japan [1]. There are many types of chemical constituents in Viburnum dilatatum (V. dilatatum), including triterpenoids, * Author to whom correspondence should be addressed. Address:School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Rd., Shenyang 110016, China; Tel.: +86-24-43520793 (Xiaoxiao Huang); +86-24-43520707 (ShaojiangSong);E-mail:*******************(XiaoxiaoHuang); ****************(ShaojiangSong).Received: 2021-04-16 Accepted: 2022-08-28phenolic glycosides and norisoprenoids [2-4]. The leaves have been utilized as a traditional Chinese medicine, and phenolic compounds have been reported as the main active chemical component of the leaves. Many researchers have analyzed the functions of these medicinal components and found that these components have good antioxidant antihyperglycemic and antiulcer effects. For example, the gamazumi crude extract obtained from the squeezed juice of the fruit prevented oxidative injury in rats [5]. This review described the chemical structures and pharmacological activities of V. dilatatum, so as to help readers understand comprehensively the research progress of V. dilatatum and provide help for the development of V. dilatatum.2 Chemical constituents and structuresPrevious reports have indicated that the main chemical constituents of V. dilatatum are phenolic glycosides and triterpenoids.2.1 Phenolic glycosidesThirteen phenolic glycosides were isolated and identified from V. dilatatum by extensive spectroscopic methods, namely p -hydroxyphenyl-6-O -trans-caffeoyl-β-D -glucoside (1) [6], p -hydroxyphenyl-6-O -trans-caffeoyl-β-D -alloside (2) [6], 4-allyl-2-methoxyphenyl-6-O -β-D -apiosyl(1→6)-β-D -glucoside (3) [6], 1-(4’-hydroxy-3’-methoxypheny1)-2-[2’’-hydroxy-4’’-(3’’’-hydroxypropyl)]-1,3-propanediol-l-O -β-D -glucopyranoside (erythro isomer) (4-7) [7], neochlorogenic acid methyl ester (8-9) [7], cryptochlorogenic acid methyl ester (10-11) [7], cyanidin-3-sambubioside (Cy-3-sam) (12) [8], cyanidin-3-glucoside (Cy-3-glc) (13) [8], 5-O -caffeoyl-4-methoxyl quinic acid (4-MeO-5-CQA) (14) [8], chlorogenic acid (5-CQA) (15) [8], quercetin (16) [8], 2-(glucopyranosyloxy)-benzyl-3-(glucopyranosyloxy)-benzoate (17) [9] and jiamizioside E (18) [10]. These structures are shown in Fig. 1.Fig. 1 Phenolic glycosides isolated from V . dilatatumContinued fig. 12.2 TriterpenoidsThere were about seventeen triterpenoids isolated and characterized from V. dilatatum , such as viburnols A (19) [11], viburnols B (20) [11], viburnols C (21) [11], viburnols D (22) [11], viburnols E (23) [11], viburnols F (24) [12], viburnols G (25) [12], viburnols H (26) [12], viburnols I (27) [12], viburnols J (28) [12],viburnols K (29) [12], viburnudienone B 2methyl ester (30) [13], viburnenone H 2 (31) [13],v i b u r n e n o n e B 2 m e t h y l e s t e r (32) [13], viburnudienone B 1 methyl ester (33) [13], viburnenone H 1 (34) [13], and viburnenone B 2 methyl ester (35) [13]. The structures are shown in Fig. 2.Continued fig. 23 Pharmacological activities3.1 Antioxidant activityOxidative stress caused by free radicals and their derivatives leads to disturbances in redox homeostasis. Reactive oxygen species (ROS) are not only endogenously produced during intracellular metabolic processes but also generated by exogenous stimuli such as UV radiation, pollutants, smoke and drugs. The cell triggers its defense systems or undergoes apoptosis when intracellular oxidative status increases. It influences numerous cellular processes including core signaling pathways, which are associated with development of systematic and chronic disorders, such as aging and cancer. Therefore, it is critical to remove cellular oxidants and restore redox balance.solution of V. dilatatum (GSS) had strong antioxidant activity in vivo and prevent stress-induced oxidative damage by the XYZ-dish method and the澳electron spin resonance (ESR) method [14]. The experimental result showed that the concentrations of lipid peroxide in plasma, liver and stomach in the GSS group were reduced. Furthermore, the activities of plasma lactic dehydrogenase, amylase and creatine phosphokinase are ordinarily increased by stress. However, these activities in the GSS group decreased to that in the control group. It was concluded that gastric ulcer formation, increase of lipid peroxidation in plasma and tissues and elevation of plasma enzymatic activities were confirmed in rats with water immersion restraint stress. It was also found that intake of GSS could protect the stomach and other tissues from oxidative damage.Kim et al. identified and isolated two major anthocyanins by NMR and LC-ESI-MS/MS, namely, cyanidin 3-sambubioside (I) and kuromanin (II) [15]. By the electron spin resonance method, the superoxide anion radical scavenging activities of I and II were evaluated with the IC 50 values of 17.3 and 69.6 µM, and their activities on hydroxyl radicals were evaluated with the IC 50 values of 4.3 and 53.2 mM. As the positive control, the IC 50 values of ascorbic acid were 74.2 µM on superoxide anion radicals and 3.0 mM on hydroxyl radicals, respectively. The above results suggested that these anthocyanins with radical scavenging properties might be the key compounds contributing to the antioxidant activity and physiological effects of V . dilatatum fruits.Woo et al. determined the free radical scavenging capacity of VD (the leaves of V. dilatatum ) [16]. Anti-oxidant activity of the extracts was assessed by the ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) or 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals. Butylated hydroxytoluene (BHT), a synthetic antioxidant, or α-tocopherol, was used as the positive control in these assays. The experimental result showed that VD inducedincrease in radical scavenging activity. In addition, lipid peroxidation inhibitory activity was determined via measurement of MDA (Malondialdehyde) levels using mouse liver tissue homogenate treated with various concentrations of the extracts. The concentration-dependent decrease in MDA levels observed was consistent with radical scavenging activities of the extracts. To examine whether VD extracts could protect mam-malian cells from oxidative stress, cultures of a human mammary gland-derived epithelial cell line MCF-7 were treated with each extract prior to challenging them with tBHP. The intracellular ROS (Reactive oxygen species) production was determined with the relative intensity of dichlorofluorescein fluorescence. While intracellular ROS formation was significantly promoted by tBHP treatment, the augmented ROS level was significantly reduced after the treatment with VD extracts.3.2 Antihyperglycemic effectIwai et al. used an oral glucose tolerance test on the diabetic rats [17]. They found that the elevation of plasma glucose level after oral administration of 2 g/kg glucose was suppressed by the repeated administration of the freeze-dried powder of V. dilatatum fruit juice (CEV). The α-glucosidase inhibitory activities of isolated compounds from CEV were also measured. Cyanidin 3-sambubioside and 5-caffeoyl quinic acid A showed inhibitory activity. These results suggested that V. dilatatum fruit had the antihyperglycemic effects.4 ConclusionV. dilatatum is distributed widely in the hills of northern China and Japan. Currently, the studies on V. dilatatum have been conducted at home and abroad, but few studies focus on its chemical components and pharmacological activities. Previousphytochemical investigations showed that the constituents of V. dilatatum included triterpenoids, phenolic glycosides, norisoprenoids and other compounds. This study describes thirteen phenolic glycosides and seventeen triterpenoids and their different degrees of antihyperglycemic, antioxidant activity and antiulcer effects, aiming to provide a reference for further studies on V. dilatatum and pharmaceutical development.References[1] Jeffrey B, Harborne A. Colour atlas of medicinal plantsof Japan. Phytochemistry, 1981, 20: 1467.[2] Miyazawa M, Hashidume S, Takahashi T, et al. Aromaevaluation of gamazumi (Viburnum dilatatum) by aroma extract dilution analysis and odour activity value.Phytochem Anal, 2012, 23: 208-213.[3] Kurihara T, Kikuchi M. Studies on the constituentsof flowers. IV. On the components of the flower of Viburnum dilatatum Thunb. J Health Sci, 1975, 95: 1098-1102.[4] Machida K, Kikuchi M. Norisoprenoids from Viburnumdilatatum. Phytochemistry, 1996, 41: 1333-1336. [5] Iwai K, Onodera A, Matsue H. Mechanism of preventiveaction of Viburnum dilatatum Thunb (gamazumi) crude extract on oxidative damage in rats subjected to stress. J Sci Food Agric, 2010, 83: 1593-1599.[6] Machida K, Nakano Y, Kikuchi M. Phenolic glycosidesfrom Viburnum dilatatum. Phytochemistry, 1991, 30: 2013-2014.[7] Machida K, Kikuchi M. Phenolic compounds fromViburnum dilatatum. Phytochemistry, 1992, 31: 3654-3656.[8] Kim MY, Iwai K, Matsue H. Phenolic compositions ofViburnum dilatatum Thunb. fruits and their antiradical properties. J Food Compos Anal, 2005, 18: 789-802. [9] Lu D, Yao S. Phenolic glycoside from the roots ofViburnum dilatatum. Nat Prod Commun, 2009, 4: 945-946.[10] Wu B, Zeng X, Zhang Y. New metabolite fromViburnum dilatatum. Nat Prod Commun, 2010, 5: 1097-1098.[11] Machida K, Kikuchi M. Viburnols: Novel triterpenoidswith a rearranged dammarane skeleton from Viburnum dilatatum. Tetrahedron Lett, 1996, 37: 4157-4160. [12] Machida K, Kikuchi M. Viburnols: Six noveltriterpenoids from Viburnum dilatatum. Tetrahedron Lett, 1997, 38: 571-574.[13] Machida K, Kikuchi M. Studies on the Constituents ofViburnum Species. XIX. Six New Triterpenoids from Viburnum dilatatum Thunb. Chem Pharm Bull, 1999, 47: 692-694.[14] Iwai K, Onodera A, Matsue H, et al. Antioxidant activityand inhibitory effect of Gamazumi (Viburnum dilatatum THUNB.) on oxidative damage induced by water immersion restraint stress in rats. Int J. Food Sci Nutr, 2001, 52: 443-451.[15] Kim MY, Iwai K, Onodera A, et al. Identification andAntiradical Properties of Anthocyanins in Fruits of Viburnum dilatatum Thunb. J Agric Food Chem, 2003, 51: 6173-6177.[16] Woo YJ, Lee HJ, Jeong YS, et al. Antioxidant Potentialof Selected Korean Edible Plant Extracts. Bio Med Res Int, 2017, 2017: 1-9.[17] Iwai K, Kim MY, Akio O, et al. Alpha-glucosidaseinhibitory and antihyperglycemic effects of polyphenols in the fruit of Viburnum dilatatum Thunb. J Agric Food Chem, 2006, 54: 4588-4592.。
Protein Expression and purification
Protein Expression and Purification22,159–164(2001)160SCOTT A.LESLEYTABLE1Genomic versus Proteomic TechnologiesGenomic technologies(DNA)Proteomic technologies(protein) Identification Determined experimentally,bioinformatics Predicted from genomic informationFunction1-dimensional information storage3-dimensional organization of chemicalfunctionalitiesBuilding blocks4bases20ϩamino acidsDetection sensitivity PCR amplification techniques Direct detection methodsSynthetic approaches Cheap and efficient oligonucleotide synthesis Limited capacity of peptide synthesismethods combined with PCRSequence determination500–700bases common by automated sequencing Direct sequencing difficult,mass spectrometry Purification Generic methods Generic methods require modification of proteinthrough gene fusionAnalysis methods Typically employ enzymes,hybridization Chemical,biophysical,biochemicaland mutagenesis),interactions between proteins(two-are of primary interest and typically are expressed in hybrid),and global protein changes(2D gels and LC–a bacterial host.Often this approach leads to problems MS).Purified protein is often required in these studies associated with expression levels and proper folding of and defines the outputs of any parallel expression and the protein of interest.Flexibility in expression options purification process.is a key parameter.Pichia or baculovirus expressionsystems can offer effective alternatives to bacterial sys-tems.Each expression scenario requires a specific vec-GENE CLONING FOR EXPRESSIONtor.Recloning cDNAs into each of these specific vectors Determining gene function through genomics typi-is extremely labor-intensive.Recombinatorial cloning cally starts from a query of a database.Sequence infor-methods provide an opportunity to minimize the effort mation for the3.9billion bases of sequence from the required for alternate expression.human genome is now available(1,2).Access and inter-Two systems are commonly used for recombinatorial pretation of this information often require sophisticated cloning and shown in Fig.1.The cre–lox recombination bioinformatics software outside the scope of this dis-system described by Elledge utilizes a single recombina-cussion.Public archives such as the Unigene Data-tion to introduce the gene of interest into a recipient base(/)or TIGR(http://vector(3).This is an in vitro reaction combined with a /)provide bioinformatic access to many in-genetic selection for the recombinant vector.In this way teresting plete genomic sequence infor-the gene of interest is cloned once into a donor vector mation is now available through Celera(http://and can then be moved into any of a number of recipient /index.cfm).Future annotation of fullplasmids for expression in different hosts or to utilize sequence information will greatly expand the access todifferent purification tags.A similar system utilizes full-length cDNA sequences.The first requirement is converting this genomic in-lambda Int/Xis/IHF recombination at att sites(4)to formation into an actual cDNA clone of that gene.Am-achieve transfer of open reading frames(ORFs).This plification of full-length cDNAs via PCR is the typical system has the advantage of a precise ORF transfer to first step.Both reverse transcriptase and amplification the expression vector rather than the cointegrant vector polymerases typically are lacking in proofreading activ-product of the cre–lox system.With either system,the ity.Care must be taken to limit the number of steps of primary limitation is that translational fusion of the amplification and to use proofreading enzymes where recombination sites is typically required to maintain possible to minimize the probability of introducing un-the flexibility and utility of the recombination method wanted mutations.Tissue selection for cDNA librariesfor expression.In those cases,such as crystallography, also is an important consideration for attempting towhere translational fusions are potentially detrimental isolate genes as they must be expressed within thatto the protein,a conventional cloning approach is library source for successful amplification.This infor-more appropriate.mation is often obtained from cDNA or oligonucleotideRegardless of the cloning method,parallel expression expression arrays.and purification requires utilization of purification Amplified gene products are cloned into appropriatetags.Many options exist for this purpose.A comprehen-vectors for expression.Depending on the source of thesive review is beyond the scope of this article.A list of gene,the host,and the end use of the protein,manydifferent vectors may be appropriate.Eukaryotic genes some commonly used tags is shown in Table2.By farHIGH-THROUGHPUT PROTEOMICS161FIG.1.Strategies of recombinatorial cloning.Individual cDNAs are cloned into a donor vector that can then be recombined into any number of recipient vectors through recombinatorial cloning.One option is to form a cointegrant plasmid through Cre-mediated recombination across a lox site.In the second scenario,a cDNA is flanked by phage lambda att sites which direct recombination into an expression vector through the use of the INT/XIS/IHF proteins.In these ways,a single donor clone can easily be transferred into any number of recipient vectors. the most common fusion is the histidine tag for purifica-analysis studies into small-scale for analysis of expres-tion on metal-chelate resins.This tag provides a sub-sion levels and properties and into large-scale for use stantial purification handle while being relatively un-with many of the proteomic applications.obtrusive as a fusion partner.Beyond purification,Small-scale expression is most useful for identifying translational fusions often provide a means to enhance those clones which express recombinant protein to high expression.The larger fusion tags such as thioredoxin levels and for evaluating the folding state of the protein. and GST often are superior in this respect.Crude expression testing is typically done by simpleSDS–PAGE analysis of whole cells.Evaluation of thefolding state is typically done by centrifugal fraction-RECOMBINANT EXPRESSION OF PROTEINation of a lysate,requiring a more gentle lysis proce-dure.Several lysozyme and mild detergent methods are Bacterial expression is most common for recombinantcommercially available for this purpose.proteins because of its ease of use and the high levelsof protein obtained.It is useful to divide expression For large-scale production for many applications,TABLE2Common Purification TagsBasis of purification Elution Reference Small tagsHistidine tag Metal affinity resin Imidizole(5)S-tag Interaction with S-protein Temperature(6) Calmodulin-binding protein Interaction with Calmodulin Calcium(7) Large tagsGlutathione S-transferase Glutathione agarose Reduced glutathione(8) Thioredoxin Phenylarsine oxide resin-Mercaptoethanol(9) Biotinylation domain Monomeric avidin resin Biotin(10) Maltose binding protein Amylose resin Maltose(11) Chitin binding protein/intein Chitin affinity Thiol(12)162SCOTT A.LESLEYtens of milligrams of protein typically are required.PURIFICATION STRATEGYEven with common bacterial expression levels,500–Proteins are highly diverse in their properties mak-1000ml of culture typically is required to provide these ing generic methods of purification difficult.Purifica-amounts.While such methods are commonplace in labo-tion tags such as described in Table2are a typical ratories,systems for parallel processing large numbers solution for purifying proteins in parallel.His-tag fu-of cultures at this level are not commercially available.sions are very common and provide a single-step chro-By developing instrumentation and optimizing media matographic purification that yields protein of suffi-and aeration conditions for high-density cell growth,cient purity for most applications.In addition,the his-our laboratory can parallel process96cultures at this tag sequence requires the addition of only six amino scale.Optical density(OD)values at600nm can reach acids to the recombinant protein,reducing the likeli-a value of40with logarithmic growth through at least hood that such a fusion will adversely affect gene func-30OD units.These cell densities allow us to producetion.A typical purification strategy is outlined in Fig.2. sufficient cell mass with65-ml culture volumes to yieldtens of milligrams of recombinant protein,sufficient for PURIFICATION AUTOMATIONmost applications.Such instrumentation is not com-Parallel processing typically involves instrumenta-monly available,but common shaking incubators cantion for automation.Lysis methods such as sonication or substitute with larger volumes.using a French press are not simple automation tasks. Recombinant expression of proteins is achievedLikewise,centrifugation is not easily integrated into through induction of a strong promoter system.Manyautomation due to problems of locating and indexing options exist in this regard including tac,T7,lambdathe rotor position.Automation of this process typically P L,and ara B promoters.It is important for parallelinvolves protocol modifications.This can easily be processing that growth and induction characteristicsachieved in small-scale methods.are consistent.For this reason,it is important to retainOn small-scale,parallel processing usually involves tight repression of expression and have a simple induc-use of a96-well plate format.Lysis is typically achieved tion procedure for high-level expression.For T7sys-using a combination of lysozyme and freeze–thaw cy-tems,the lac operator and T7lysozyme(pLysS)provide cles.Phage lysozymes are more effective than hen egg an extra level of repression.The arabinose promoter is white lysozyme for this purpose and can be combined tightly repressed in the absence of inducer and is our with nucleases to reduce viscosity and facilitate re-preferred system for parallel growth.With all of the moval of cell debris at the low g forces commonly used promoters listed,recombinant expression levels of10–with microtiter plates.Alternatively,nonionic deter-50%of total cell protein are common.gents can be employed for nondenaturing lysis.FIG.2.Generalized purification strategy of recombinant fusion protein.A common purification strategy is shown here.Proteins are purified from fermentation cultures by affinity purification.Isolated cell pellets are resuspended in an appropriate lysis buffer and disrupted by high-intensity sonication.Cell walls and insoluble debris are pelleted by centrifugation and the soluble supernatant containing the recombinant fusion protein is applied to chromatography resin containing an immobilized metal for affinity purification.Fractions containing the recombinant protein can be used directly or further purified using conventional chromatographic techniques.HIGH-THROUGHPUT PROTEOMICS163TABLE3Robotic Systems with Capabilities Adaptable to ProteinPurificationManu-facturer Instrument WebsiteQiagen BioRobot3000/Tomtec Quadra96/Matrix PlateMate /Hamilton Microlab4200/Beckman Biomek2000/Packard PlateTrak /index2.htmGilson Nebula215/index.htmlRobotic systems for nucleic acid purification are rela-tively commonplace and have recently been adapted forprotein purification.The Qiagen BioRobot3000per-forms multiple functions relevant to protein purifica-tion.It provides aspirate,dispense,pipet,vacuum fil-tration,and plate-shaking functions on a relativelycompact platform.These functions can be adapted to FIG.4.SDS–PAGE analysis of purified protein.Metal affinity chro-perform cell lysis and chromatography steps from1–2matography yields highly purified protein from a single chromato-graphic separation.This gel shows typical yields and purity obtained ml of bacterial culture.Specialized96-well plates clearfrom parallel purification using an automated purification system. cell debris via vacuum filtration and are also used toSuch proteins have been incorporated directly in successful crystalli-retain resin for chromatographic separations.The Wal-zation trials.Ten-microliter samples of12ml protein eluates from Ni-lac Quadra96also has most of these capacities and resin were separated by10%SDS–PAGE.Samples are recombinant can parallel process96or384samples.Both of thesefusions of thioredoxin to human proteins as indicated by accessionnumber.systems have been used with success in our laboratoryfor small-scale protein purification of proteins in micro-titer plates.Table3lists some robotic systems that maybe applied to small-scale protein purification.providing the throughput needed for proteomic studies Despite the difficulties,large-scale protein purifica-involving tens of thousands of proteins.We are cur-tion also can be automated.In our laboratory we simul-rently able to process approximately96–192proteins taneously process96bacterial cultures of65–70ml.per day with this system with average yields of around Instrumentation for processing96parallel cultures at10mg of purified protein.Affinity purification results that scale required development of custom roboticsin recombinant protein that is typically80–90%pure shown in Fig.3.These robotics incorporate liquid aspi-(see Fig.4)which is sufficient for most applications. rate and dispense,centrifugation,and sonication capa-Subsequent purification is sometimes necessary,for ex-bilities required for purification.Automation is key to ample,in protein crystallography,and is achieved usingFIG.3.Protein purification automation.Custom robotics for performing the purification strategy outlined in Fig.2are shown.(a)The instrument has capacity for automated liquid aspiration and dispensing,sonication,centrifugation,and fractionation.Ninety-six cultures are processed in parallel,giving up to10–50mg of purified protein per culture.(b)Expanded view of aspirate/dispense/sonicate head accessing rotor.164SCOTT A.LESLEYREFERENCESstandard ion-exchange and size-exclusion chromatog-raphy.Automation of these methods is relatively1.Venter,J.C.et al.(2001)The sequence of the human genome. straightforward employing standard FPLC and au-Science291,1304–1351.tosampler instrumentation.2.International Human Genome Sequencing Consortium(2001)Initial sequencing and analysis of the human genome.Nature SUMMARY409,860–921.3.Liu,Q.,Li,M.Z.,Leibham,D.,Cortez,D.,and Elledge,S.J. Determining gene function and understanding the(1999)The univector plasmid-fusion system,a method for rapid relationships and interactions of the gene products are construction of recombinant DNA without restriction enzymes.a global effort in biological studies.The approach toCurr.Biol.8,1300–1309.performing this immense task is driven by the availabil- 4.Hatley,J.L.,Temple,G.F.,and Brasch,M.A.(2000)DNA cloningity of genomic information.To utilize this informationusing in vivo site-specific recombination.Genome Res.10,pp.1788–1795.for experimentation,however,significant effort isneeded to actually isolate and express proteins from5.Petty,K.J.(1996)Metal-chelate affinity chromatography,in“Current Protocols in Molecular Biology,”Vol.2,Wiley,New York. the genes of interest for study.The complexity of this6.Kim,J.S.,and Raines,R.T.(1993)Ribonuclease S-peptide as a effort is compounded by the large number of gene prod-carrier in fusion proteins.Protein Sci.2,348–356.ucts comprising the proteome.Parallel processing and7.Stofko-Hahn,R.E.,.Carr,D.W,and Scott,J.D.(1992)A single generic methods are required to achieve a systematicstep purification for recombinant proteins.FEBS Lett.302, and thorough evaluation of gene function.274–278.Experimental uses of proteins for structural and8.Smith,D.B.,and Johnson,K.S.(1988)Single-step purification functional studies typically require milligram amounts of polypeptides expressed in Escherichia coli as fusions within purified form.Unlike genomic technologies that pri-glutathione S-transferase.Gene67,31–40.marily involve the study of nucleic acids,proteomic9.Lu,Z.,DiBlasio-Smith,E.A.,Grant,K.L.,Warne,N.W.,LaVallie,studies focus on proteins.Proteins are by nature much E.R.,Collins-Racie,L.A.,Follettie,M.T.,Williamson,M.J.,more diverse in composition and properties than nucleicand McCoy,J.M.(1996)Histidine patch thioredoxins.Mutantforms of thioredoxin with metal chelating affinity that provide acids.In many ways,this makes them more interestingfor convenient purifications of thioredoxin fusion proteins.J. but also less amenable to generic methods and technolo-Biol.Chem.271,5059–5065.gies for parallel processing.Nonetheless,methods and10.Cronan,J.E.(1990)Biotination of proteins in vivo.A post-trans-instrumentation are currently available to meet this lational modification to label,purify,and study proteins.J.Biol.ing these advances will allow a systematic Chem.265,10327–33.effort at understanding biological pathways at the11.Maina,C.V.,Riggs,P.D.,Grandea,A.G.,Slatko,B.E.,Moran,molecular level.L.S.,Tagliamonte,J.A.,McReynolds,L.A.,and Guan,C.D.(1988)An Escherichia coli vector to express and purify foreign ACKNOWLEDGMENTS proteins by fusion to and separation from maltose-binding pro-tein.Gene74,365–373.The author acknowledges the help of Marc Nasoff,Heath Klock,Dan McMullan,Tanya Shin,Juli Vincent,Mike Hornsby,Mark12.Chong S.,Mersha F.B.,Comb D.G.,Scott M.E.,Landry D.,Vence L.M.,Perler F.B.,Benner J.,Kucera R.B.,Hirvonen C. Knuth,Loren Miraglia,and Jeremiah Gilmore for their contributionsto the high-throughput cloning and expression efforts.He also recog- A.,Pelletier J.J.,Paulus H.,and Xu M.Q.(1997)Single-column nizes Bob Downs,Mark Weselak,Andy Meyer,and Jim Mainquistpurification of free recombinant proteins using a self-cleavable and the rest of the GNF engineering staff for their contributions to affinity tag derived from a protein splicing element.Gene192, the custom robotics that make this effort possible.271–281.。
DNA的复制课件-2023-2024学年高一下学期生物人教版(2019)必修2
C.这M个子代T₂噬菌体中，含32P的T₂噬菌体所占的比例为1/M
有2个，故其所占的比例为2/M,C 错误。培养足够长的时间，会出现 不含32P而含35S的T₂噬菌体，但一般不会出现含32P的大肠杆菌，D 错 误。
3 、在一个密闭的容器里，用含有同位素13C的脱氧核苷酸合成一个 DNA分子，然后加入普通的含12C的脱氧核苷酸，经n次复制后，所得DNA 分子中含¹2C的脱氧核苷酸链数与含13C的脱氧核苷酸链数之比是
特点 半保留复制；边解旋边复制；多起点复制；双向复制
结果 子链与母链结合，构成两个相同的新的DNA分子
意义 保持了遗传信息的连续性
总结规律
规律1:若 一 个DNA 复制n次。
1.在子代中共形成 2n 个DNA, 其中含有亲代
DNA链的DNA分子数 2 0 2.含有亲代DNA链的DNA分子数占DNA分子总
D . 含 1 0 0 个 碱 基 对 ( 其 中 胞 嘧 啶 6 0 个 ) 的 DNA复 制 3 次 共 需 要 2 8 0 个
2、用 DNA双链均被32P标记的一个T,噬菌体侵染被35S标记的大肠杆菌， 一段时间后释放出出了M个子代T₂噬菌体。下列有关叙述正确的是
()
A.用32P标记T₂噬菌体的方法与用35S标记大肠杆菌的方法相同
It has not eseaped our notice that the specife pairing we have postulated immediately guggests a p⁰ssible copying mechanism for the genetic materiaI.
Full details of the structure,including the conditions assumed in building it,together with a set of co-ordinatos for the atoms,will be published elsewhere.
高三英语询问科学单选题50题
高三英语询问科学单选题50题1. Recent research has found that some bacteria can form a complex community structure called a biofilm. In a biofilm, bacteria are surrounded by a self - produced matrix. Which of the following is a major component of this matrix?A. DNAB. ProteinC. LipidD. Carbohydrate答案：D。
解析：在生物膜的基质中，碳水化合物是主要成分之一。
选项A，DNA虽然存在于细胞中，但不是生物膜基质的主要成分。
选项B，蛋白质是生物膜的组成部分，但不是基质的主要成分。
选项C，脂质主要参与细胞膜结构构建，而非生物膜基质的主要部分。
本题主要考查生物科学知识，语法上是一般现在时的陈述语句。
2. The mitochondria are known as the "powerhouses" of the cell. Which process mainly occurs in mitochondria?A. PhotosynthesisB. GlycolysisC. Cellular respirationD. Protein synthesis答案：C。
解析：线粒体中主要发生的过程是细胞呼吸，这是其重要功能。
选项A，光合作用主要发生在叶绿体中。
选项B，糖酵解发生在细胞质中。
选项D，蛋白质合成主要发生在核糖体上。
从语法来看，这是一个考查一般现在时和生物知识结合的题目。
3. In the process of evolution, some animals have developed unique adaptations. The giraffe's long neck is an example. Which theory best explains the evolution of the giraffe's long neck?A. Lamarck's theory of inheritance of acquired characteristicsB. Darwin's theory of natural selectionC. Mendel's law of inheritanceD. The theory of punctuated equilibrium答案：B。
211091445_牡丹籽粕蛋白提取工艺优化和功能性质分析
李若敏，张焕新，盘赛昆，等. 牡丹籽粕蛋白提取工艺优化和功能性质分析[J]. 食品工业科技，2023，44（8）：197−204. doi:10.13386/j.issn1002-0306.2022050251LI Ruomin, ZHANG Huanxin, PAN Saikun, et al. Process Optimization and Functional Properties of Peony Seeds Protein[J]. Science and Technology of Food Industry, 2023, 44(8): 197−204. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022050251· 工艺技术 ·牡丹籽粕蛋白提取工艺优化和功能性质分析李若敏1,2，张焕新1, *，盘赛昆2，叶静静1（1.江苏农牧科技职业学院，江苏泰州 225300；2.江苏海洋大学食品科学与工程学院，江苏连云港 222005）摘 要：以酶水解-超声辅助碱溶酸沉法提取蛋白工艺为基础，初步对牡丹籽中粗蛋白进行分离提取。
通过单因素实验和响应面试验，考察料液比、超声温度、酶用剂量、超声时间四个因素对牡丹籽粕蛋白提取率的影响，确定最佳提取工艺，并测定其功能特性。
结果表明，酶水解-超声辅助碱溶酸沉法提取牡丹籽粕蛋白最优工艺条件为：料液比为1:9.8（w/v ），超声温度为49.5 ℃，酶用剂量为1.9%，超声时间为119 min 。
在此条件下，蛋白质提取率达到90.95%。
此时所得蛋白与常规法提取蛋白相比，氨基酸种类齐全、必需氨基酸含量均有所提高，功能特性如持水性、吸油性、乳化性皆优于常规法提取蛋白的功能特性，且乳化的稳定性更优，由此推测可作为食品加工乳化剂。
因此酶水解-超声辅助碱溶酸沉法提取的牡丹籽粕蛋白具有更高的营养价值和更好的功能特性。
【必刷题】2024高一英语上册英语学术论文阅读专项专题训练(含答案)
【必刷题】2024高一英语上册英语学术论文阅读专项专题训练（含答案）试题部分一、选择题：1. 在英语学术论文中，以下哪个部分通常用来概述研究背景和目的？A. IntroductionB. MethodologyC. ConclusionD. Literature Review2. 以下哪个词组常用于表示“结果表明”？A. As a resultB. It turns out thatC. The findings show thatD. In conclusion3. 在阅读英语学术论文时，以下哪个部分可以帮助我们了解研究方法？A. AbstractB. IntroductionC. MethodologyD. Discussion4. 以下哪个词组表示“在某种程度上”？A. To some extentB. In some wayC. On the other handD. As a matter of fact5. 在学术论文中，以下哪个标点符号用于引号内的句子结尾？A. CommaB. PeriodC. Question markD. Exclamation point6. 以下哪个词组表示“”？A. In additionB. HoweverC. ThereforeD. Moreover7. 在阅读学术论文时，以下哪个部分可以帮助我们了解研究的主要发现？A. IntroductionB. MethodologyC. ResultsD. Literature Review8. 以下哪个词组表示“尽管如此”？A. NeverthelessB. ThereforeC. MoreoverD. Otherwise9. 在学术论文中，以下哪个部分通常用于提出研究问题？A. IntroductionB. MethodologyC. DiscussionD. Conclusion10. 以下哪个词组表示“考虑到”？A. Taking into accountB. Given thatC. In order toD. On the condition that二、判断题：1. 学术论文的应简洁明了，直接反映研究主题。
新教材同步备课2024春高中生物第3章基因的本质3.3DNA的复制课件新人教版必修2
(2)注意碱基的单位是“对”还是“个”。 (3)切记在DNA复制过程中，无论复制了几次，含有亲代脱氧 核苷酸单链的DNA分子都只有两个。 (4)看清试题中问的是“DNA分子数”还是“链数”，“含” 还是“只含”等关键词，以免掉进陷阱。
二、DNA分子的复制
例1.某DNA分子中含有1 000个碱基对(被32P标记)，其中有胸腺 嘧啶400个。若将该DNA分子放在只含被31P标记的脱氧核苷酸的 培养液中让其复制两次，子代DNA分子相对分子质量平均比原来 减少 1 500 。
F2:
提出DNA离心
高密度带 低密度带 高密度带
低密度带 高密度带
一、DNA复制的推测—— 假说-演绎法
1.提出问题 2.提出假说
（1）演绎推理 ③分散复制
15N 15N
提出DNA离心
P:
3.验证假说
15N 14N
F1:
细胞分 裂一次
转移到含 14NH4Cl的培养 液中
提出DNA离心
细胞再 分裂一次
二、DNA分子的复制
例3.若亲代DNA分子经过诱变，某位点上一个正常碱基变成了5-溴 尿嘧啶（BU），诱变后的DNA分子连续进行2次复制，得到4个子 代DNA分子如图所示，则BU替换的碱基可能是（ C ）
A.腺嘌呤 C.胞嘧啶
B.胸腺嘧啶或腺嘌呤 D.鸟嘌呤或胞嘧啶
二、DNA分子的复制
例4. 5-BrU(5-溴尿嘧啶)既可以与A配对，又可以与C配对。将一 个正常的具有分裂能力的细胞，接种到含有A、G、C、T、5-BrU 五种核苷酸的适宜培养基上，至少需要经过几次复制后，才能实现 细胞中某DNA分子某位点上碱基对从T—A到G—C的替换（ B ）
胃癌细胞 herg mRNA、HERG 蛋白表达及 HERG 电流强度变化
胃癌细胞 herg mRNA、HERG 蛋白表达及 HERG 电流强度变化邵晓冬;张永国;陈江;王金玲;郭晓钟;任丽楠【摘要】目的：探讨胃癌细胞herg mRNA、HERG蛋白表达及HERG电流强度的变化的临床意义。
方法培养胃癌细胞（胃癌细胞系SGC7901、MGC803、AGS、MKN45）及永生化胃上皮细胞（ GES ），取对数生长期细胞用于实验。
采用RT-PCR法检测herg mRNA表达，Western blot法检测HERG蛋白表达，采用全细胞膜片钳技术测定SGC7901及GES的HERG电流强度。
结果 herg mRNA及其蛋白在4种胃癌细胞系中均有表达，AGS中HERG蛋白表达量低于其他三种细胞系（ P均＜0．05）；GES中无herg mRNA及其蛋白表达。
在SGC7901中检测到HERG电流， GES中未记录到HERG电流。
结论 herg mRNA及其蛋白在胃癌细胞系SGC7901、MGC803、AGS、MKN45表达增高， SGC7901中存在HERG电流。
HERG蛋白可能参与了胃癌的发生，并与胃癌恶性程度有关。
%Objective To investigate the expression of herg gene , HERG protein and HERG current in gastric cancer cells.Methods Gastric cancer cells and gastric epithelial ( GES) cells were cultured to logarithmic phase .The expres-sions of herg mRNA and HERG protein in gastric cancer cells and GES cells were measured by using RT -PCR and Western blot, respectively.The whole cell configuration of the patch-clamp techniquewas employed to record HERG currents in va-rious cells .Results HERG mRNA and protein were positively expressed in four gastric cancer cell lines .Expression level of HERG protein in AGS cells was lower compared with the other three gastric cancer cell lines (P<0.05).There was negativeexpression of herg mRNA and HERG protein in GES cell line .HERG current was detected in gastric cancer cell , whereas there was no HERG current in GES cell .Conclusion Herg gene and HERG protein were highly expressed in gas-tric cancer cells and HERG current was exclusively detected in gastric cancer cells .HERG protein was associated with car-cinogenesis of gastric cancer and may serve as a diagnostic marker for gastric cancer .【期刊名称】《山东医药》【年(卷),期】2015(000)002【总页数】4页(P1-4)【关键词】胃肿瘤,胃癌;herg基因;HERG蛋白;HERG电流;延迟整流钾通道;肿瘤形成过程【作者】邵晓冬;张永国;陈江;王金玲;郭晓钟;任丽楠【作者单位】沈阳军区总医院，沈阳110016;沈阳军区总医院，沈阳110016;沈阳军区总医院，沈阳110016;沈阳军区总医院，沈阳110016;沈阳军区总医院，沈阳110016;沈阳军区总医院，沈阳110016【正文语种】中文【中图分类】R735.2·论著·胃癌的发病机制目前尚未清楚，化疗和生物治疗效果不理想。
Protein__Expression_of_Proteins_in_E.coli
Expression of Proteins in E. coliExpression of a recombinant protein can beapproached in general by constructing a plasmid thatencodes the desired protein, introducing the plasmidinto the required host cell, growing the host cells andinducing protein expression, and then lysing the cells,purifying the protein, and performing SDS-PAGEanalysis to verify the presence of the protein (Figure 1).The protocols and recommendations given in thePlasmid DNA chapter for the handling andtransformation of E. coli are also valid for theproduction of recombinant proteins. With carefulchoice of host strains, vectors, and growth conditions,most recombinant proteins can be cloned andexpressed at high levels in E. coli.Optimal growth and expression conditions for theprotein of interest should be established with small-scale cultures before large-scale protein purification isattempted.Basic principlesThis section discusses critical factors to be considered when expressing foreign proteins in E. coli.Culture mediaThe media of choice for the growth of E. coli cells containing an expression plasmid are LB medium and itsmodifications, 2x YT, or Super Broth, each containing the relevant selective antibiotic(s). Initially it is advisableto try expression in all three media in parallel, and to do a time course analysis to monitor growth and expressionafter induction. Striking differences between the level of expression in different media and at different times areoften observed.Maintenance of the expression plasmidPoor plasmid maintenance in the cells can lead to low expression levels. Ampicillin is an unstable antibioticand is rapidly depleted in growing cultures due in part to the β-lactamase secreted by resistant bacterial cells.It is important to check plasmid levels by plating cells from the expression culture on plates with and withoutampicillin. If the stability of the expression construct is a problem, the cultures should be grown in the presenceof 200 µg/ml ampicillin, and the level should be maintained by supplementing ampicillin during long growthperiods. Alternatively, the cultures may be grown in the presence of carbenicillin, a more stable β-lactam, at50 µg/ml (see “Antibiotics”, page 5).Figure 1.Overview of the steps involved in expression and analysisof recombinant proteins.Generation of Recombinant ProteinsSmall-scale expression culturesSmall-scale expression and purification experiments are highly recommended andshould be performed before proceeding with a large-scale preparation. In manycases aliquots of the cells can be lysed in a small volume of sample buffer andanalyzed directly by SDS-PAGE. The use of small expression cultures provides arapid way to judge the effects of varied growth conditions on expression levelsand solubility of recombinant proteins. Expression levels vary between differentcolonies of freshly transformed cells, and small-scale preparations permit theselection of clones displaying optimal expression rates.Induction of protein expressionThe method used for induction of protein expression is dependent on the plasmidvector and E. coli strain used. Protein expression can be induced by a raising ofthe incubation temperature or by the addition of an inducing chemical such asisopropyl-β-D-thiogalactoside (IPTG) to the culture medium. Details of inductionmethods and the plasmids they relate to can be found in standard molecularbiology texts (1,2).Time-course analysis of protein expressionTo optimize the expression of a given protein construct, a time-course analysis bySDS-PAGE (Protocol 5, page 75) of the level of protein expression is recommended.Intracellular protein content is often a balance between the amount of solubleprotein in the cells, the formation of inclusion bodies, and protein degradation. Bychecking the protein present at various times after induction, the optimal inductionperiod can be established (Figure 2).Colony blotsWe recommend the colony-blot procedure (Figure 3;colonies that do not express protein.Note: If using the QIA express® Expression Systemmeans that small peptides (<30 amino acids) expressed from QIAexpressing the desired protein.Figure 3.Detection of positive expression clones by colony blotting.M C0.5 h 1 h 2 h3h4hFigure 2.Time course analysis of the expression of dihydrofolatereductase (arrowed). Aliquots were removed at the times indicatedand analysed by SDS-PAGE. C: uninduced control. M: markers.➜Time-Course Analysis of Protein Expression。
红、黄、绿三种颜色荧光质粒导入大肠杆菌中的稳定性表达
2018年6月第3期193〜198甘肃农业大学学报JOURNAL OF GANSU A G R IC U LTU R A L U N IVER SITY第53卷双月刊红、黄、绿三种颜色荧光质粒导入大肠杆菌中的稳定性表达杨晓玫，师尚礼(甘肃农业大学草业学院，甘肃兰州730070)摘要:【目的】红、黄、绿3种颜色荧光蛋白质粒导人大肠杆菌的热稳定表达是实现质粒与受体菌杂交获得荧光标记菌的关键，荧光标记菌的构建，可极大地丰富荧光标记菌的颜色类别，为微生物示踪研究提供丰富的工具.【方法】采用大肠杆菌D H5a热激转导法构建荧光标记菌.【结果】黄色和绿色蛋白质粒在原液浓度基础上，稀释10倍进行热激转导，红色蛋白质粒原液直接进行热激转导，可将携带红、黄、绿3种颜色荧光蛋白的质粒D N A成功导人大肠杆菌.【结论】经PCR重复检测，条带明显并符合红、黄、绿3种颜色荧光质粒基因序列大小,3种颜色荧光质粒热稳定表达水平较高.关键词：大肠杆菌;质粒转导;荧光蛋白;基因表达中图分类号:Q78文献标志码：A文章编号：1003-4315(2018)03-0193-06Gene expression of red, yellow and green fluorescence plasmidstability after transferred in E sch erich ia coliY A N G X ia o-m e i,S H I S hang-ii(College of Pratacultural Science,Gansu Agricultural University,Lanzhou730070,China)A bs狉a c t:【O b je c iv e】T h e h e a t-sta b le e x p re s s io n o f re d,y e llo w a n d g re e n flu o re s c e n t p ro te in s a fte r tra n s fe rre d in to Escherichia coli is th e k e y to realize th e h y b rid iz a tio n o f p la o b ta in flu o re s c e n t labeled ba cte ria.T h e c o n s tru c tio n o f flu o re s c e n t labeled bacteria can g re a tly e n rich th e c o lo r o f flu o re s c e n tm a rk e r b a c te ria,and p ro v id e ab u n d a n t to o ls fo r m ic ro b ia l tra c e r research.【M e th o d】D H5a w as used to c o n s tru c t flu o re s c e n t labeled b a cte ria.【R e s u lt】Y e llo w and green p la sm id p ro te in s w ere subjected to heat shock tra n s d u c tio n w ith10 tim e s d ilu tio n,w h ile th e re d p la s m id p ro te in w as d ire c tly subje cte d to heat shock tra n s d u c tio n w ith o u t d ilu tio n.【C o n clu sio n】P C R d e te ctio n show ed th a t th e s trip s w ere obvio us and accorded w ith th e sequence sizes o f3flu o re s c e n t p la sm id s.T h e p la s m id and green w e re tra n s fe rre d in to Escherichia coli su cce ssfu lly w ith h ig h e r heat stable expression le v e l.K ey words：Escherichia coli；p la sm id tra n s d u c tio n；flu o re s c e n t p ro^t e in；gene expression荧光蛋白（flu o re s c e n t p ro te in)作为最有效的 态的观察标记细胞，起到简单又实用的示踪作用.绿活细胞标记物质，在蓝光激发下可以发荧光，无需试色荧光蛋白(G F P)最早是从维多利亚发光多管水母剂检验，实现了对细菌长期稳定的标记，可实时和动中发现并纯化得到的一种天然荧光蛋白，其不需要第一作者:杨晓玫(1992-)，女，硕士研究生，主要从事草地生物多样性的研究.E-mail:yangxiaomeirz@126. com通信作者:师尚礼，男，教授，博士生导师，主要研究牧草与草坪草育种.E-mail:2470398455@qq com基金项目：国家自然科学基金项目（1560666).收稿日期：2017-03-05;修回日期：2017-04-06194甘肃农业大学学报2018 年任何底物和辅助因子就能够在多种活体生物体内进行异源表达[1].M a t z等2从不发光的海洋生物珊瑚种群（A n th o ro a n species)克隆到 G K P基因，G F P 具有广谱表达性，可在细菌、植物、真菌、鱼类及哺乳动物中表达和应用，也可进行活细胞定时定位观察，表达后能自发产生绿色荧光[1].红色荧光蛋白 (R F P)是M a t z等21999年从印度洋和太平洋地区的珊瑚虫中分离，与绿色荧光蛋白同源的荧光蛋白，在紫外线的照射下可发射红色荧光，其最大吸收波 长为558 n m,最大发射波长为583 n m.B eachy[3]、G u id o等4用D s R e d作为植物报告基因对烟草进 行试验，发现D s R e d对植物的生长发育无负面影 响;黄色荧光蛋白（Y F P)是绿色荧光蛋白的一种突 变体，最初来源于维多利亚多管水母(Aegw orea Vict o r ia),与绿色荧光蛋白相比，荧光偏移于红色光 谱，其最大激发波长为514 n m,最大发射波长为527 n m,孙金钰[]等的研究为Y F P在生物学领域的广 泛应用提供充足的原料来源.大肠杆菌(E i/i r T/i i a c〇r)是一种常用的模式生物，其信号转导方面和其他原核生物以及真核生 物相似，如今已经被广泛应用于细菌的转导机制研 究，成为研究较为广泛的模式菌[].自从进入基因时代后，因大肠杆菌的性质，其作为辅助菌研究基因、蛋白质等的首选材料，揭示了很多基因表达的未知 领域，为大肠杆菌表达系统提供了更广泛的发展空 间，随着生物学技术的迅猛发展，大肠杆菌在试验研究及重组蛋白应用中占据的地位将越来越重要.目前，荧光蛋白标记是检测和示踪侵染最直观、精确且易于操作的方法[7]，已有青色荧光蛋白成功构建且能稳定表达，但荧光资源短缺，一种标记荧光蛋白质粒不便于同时多元化标记.基于红、黄、绿3种荧光蛋白质粒转导大肠杆菌的研究较少，本试验 将3种荧光蛋白质粒R ed-123、Y F P-69、G F P-104热激转导于大肠杆菌中，观察导入的荧光蛋白质粒在 大肠杆菌中能否正常表达，检测红、黄、绿荧光蛋白能否转导至大肠杆菌并稳定表达，研究3种荧光蛋白质粒热激转导的不同条件及其对大肠杆菌的影 响.乐易林[]认为电击转导大肠杆菌的化学诱导剂 价格昂贵并且有些诱导剂具有毒性，不利于一些药 用蛋白的生产，因此，热激诱导外源基因是比较理想的选择，成本低、无毒性.本研究采用传统的热激转 导方法，将荧光质粒转导至大肠杆菌中.1材料与方法1.1培养基L B液体培养基和L B固体培养基.1.2试验材料试验所用红、黄、绿3种颜色蛋白质粒均由新西兰梅西大学张学贤教授赠，具氨苄青霉素抗性.1.2.1主要试剂及常用酶 E x T a gA R T M H S D N A P o ly m e ra s e,Q u ic k C u tT M S m a l，Q u ic k C u tT M X b a l，T4D N A L ig a s e,D N A M a r k-e1000均购自大连宝生物工程有限公司.抗生素 K a n a粉末、抗生素A m p粉末、P C R引物合成和样 品测序均由生工生物工程（上海）股份有限公司 完成.1.2.2质粒载体及引物1.2.2.1质粒载体及引物大肠杆菌感受态细 胞购自全式金生物技术公司，T-V e c to r P M D19 (S im ple)购自宝生物工程有限公司，红、黄、绿3种颜色引物均由生工生物工程(上海）股份有限公司 合成根据转化成功的3种颜色，保存于的30 %浓度的甘油中，随机挑选3管进行测序，用O F F D E R在线软件确定3种颜色的开放空间，获得其质粒的完 整序列.根据已获得G F P104、Y F P69、只《«23的基 因序列，P rim e rP re m ie r5软件设计上下游引物，进行测序，引物和测序均有由生工生物工程(上海）股份有限公司合成.红色R e d l23引物序列为:上游引物:5'-A T G G T G A G C G G C C T G C T G A A G G A G A-3';下游引物：5'-C T C G G G C A G G T C G C T G T A C-C G G G C C3.黄色Y F P69弓丨物序列为：上游弓丨物: 5,-G T C C G C C A T G C C C G A A G G C T A C G T C-3，；下游引物：5'-C T T G T A C A G C T C G T C C A T G C C G A G A-3'绿色G FP104引物序列为:上游引物5-A A A G-G A G A A G A A C T T T T C A C T G G A G3';下游引物：5'-G T T T G C T G C A G G C C T T T T G T A T A G-3'.1.3试验方法1.3.1红、黄、绿质粒D N A 转化大肠杆菌感受态第3期杨晓玫等:红、黄、绿三种颜色荧光质粒导入大肠杆菌中的稳定性表达195细胞取50，刚刚化冻的D H 5a 感受态细胞，分别加入1 p L 的原浓度培养13h 连接液、原浓度稀 释10倍培养13h 连接液和原浓度稀释100倍培养 13h 连接液，轻轻混勻，在冰浴中静置30m in ;42°C 水浴中热击45 s ，然后快速平稳的转移离心管于冰 浴中2m in ，此过程不要剧烈摇动离心管;然后向离 心管中加入500 p L 的常温L B 液体培养基(不含任 何抗生素），混勻，37°C 条件下，200 r /m in 摇床培养 1h ，使细胞复苏;振荡培养结束后，5 000 r /m i n 离 心2m in 左右，然后倒掉大部分上清液，留下100〜 150 PL 菌液，用移液枪轻轻吸打混勻;将混勻的菌 液均勻涂于已倒好的L B 固体平板(含A m p )，正面 朝上放置30m i n 左右，待培养基完全吸收菌液后， 倒置平板，37C 静培养12〜16h ;平板转至4C ，仍 然倒置保存.1.3.2测序选择13h 培养后的白色菌落，挑取 单菌落于L B 液体培养基中（含A m p ) 37 C 、 230 r /m in 恒温摇床培养13 h 左右，保存于30%的 甘油中，随机挑选进行测序，重复3次，双向测序成 功转至一80 C 保存，对其菌液进行P C R 扩增验证. 1.3.3目的基因的克隆1.3.3.1扩增目的片段以热激法转化的菌液为 模板，参考冯光燕的程序9进行P C R 扩增.1) 红色质粒菌液的P C R 扩增 R e d l 23P C R 扩增程序依次为：首先94 C 预变性4 m m ，然后 94 C 30 s ，50 C 〜60 C 30 s ，72 C 45 s ，进行 35 个循环，最后72 C 延伸8 m in ，转至4 C 保存产物， 待1.0%琼脂糖凝胶电泳检测.R e d l 23P C R 扩增体 系参考马玲珑的方法[10]，利用1%的凝胶凝胶电泳 检测.2) 黄色质粒菌液的P C R 扩增 Y F P 69P C R扩增程序依次为：首先94 C 预变性4 m in ，然后 94 C 30 s ，52 C 30 s ，72 C 45 s ，进行 35 个循环， 最后72 C 延伸8m in ，转至4 C 保存产物，待1.0% 琼脂糖凝胶电泳检测.Y F P 69P C R 扩增体系参考马 玲珑的方法[0]，利用1%的凝胶凝胶电泳检测.3) 绿色质粒菌液的P C R 扩增 G FP 104PCR 扩增程序依次为:首先94 C 预变性4m in ，然后94 C 30 s ，52 C 〜64 C 30 s ，72 C 45 s ，进行 35 个循环， 最后72 C 延伸8m in ，转至4 C 保存产物，待1. 0%琼 脂糖凝胶电泳检测.G FP 104P C R 扩增体系参考马玲 珑的方法[10]，利用1%的凝胶凝胶电泳检测.2结果与分析2.1不同浓度原液对红、黄、绿3种颜色蛋白质粒 的影响浓度的大小反映了荧光蛋白质粒转导能力的强 弱，黄色和绿色蛋白质粒转导能力均高于红色蛋白 质粒，其中，转导能力强的蛋白质粒因其单菌落太过 密集需稀释转导.红色蛋白质粒(R F P )原液热激转导后L B 平板 上长出单一的白色菌落(图1-A )，原液稀释10倍液 和原液稀释100倍液热激转导后L B 平板上均无单 一的白色菌落（表1)，表明红色蛋白质粒需原液热 激转导．黄色蛋白质粒(Y F P )原液热激转导后L B 平板 上长出密集的非单一白色菌落，原液稀释10倍液热 激转导后L B 平板上长出单一的白色菌落(图1-B )， 原液稀释100倍液热激转导后L B 平板上未长白色RFP YFP GFP图1红、黄、绿3种颜色蛋白质粒热激转导培养的单菌落Figure 1 Red ^yellow and green three colors plasmid heat shock protein transduction of singlecolony196甘肃农业大学学报2018 年菌落(表1)，表明黄色蛋白质粒需原液稀释10倍热激转导.绿色蛋白质粒(G F P)原液热激转导后L B平板上长出密集的非单一白色菌落，原液稀释10倍液热激转导后L B平板上长出单一的白色菌落(图1-C),原液稀释100倍液热激转导后L B平板上未长白色菌落(表1)，表明绿色蛋白质粒需原液稀释10倍热激转导.表1不同浓度下红、黄、绿3种蛋白质粒单菌落生长情况Table1Single colony growth of three kinds of plasmidproteins of red,yellow and green at different concentrations不同浓度原液单菌落生长情况颜色原液原液稀释10倍液原液稀释100倍液红色质粒十一一黄色质粒一十一绿色质粒一十+指涂板能长出白色单菌落，一表示涂板不能长出白色单菌落.2.2 P C R扩增不同温度对红、黄、绿3种颜色蛋白质粒的影响温度的不同探索了3种颜色蛋白质粒扩增复性的最适温度，黄色蛋白质粒扩增的最适温度为唯一 定值，红色和绿色蛋白质粒扩增的最适温度均为温 度区间，选择性较高.以红色质粒R e d l23过夜单菌落摇床菌液为模 板，参考使用高保真酶进行P C R，扩增结果如图2.图2-A中1一7分别表示50〜62°C条件，每隔2°C 设置1个梯度值，在该条件下复性，持续35个循环.图2-A中的50,52,54 C温度条件菌液能看到很亮 的单一条带，56C条件菌液条带较暗，58,60,62 C 条件的菌液均无条带（表2),可知红色蛋白质粒 P C R适宜温度为50〜54C.以黄色质粒Y F P69过夜单菌落摇床菌液为模板，使用高保真酶进行P C R,扩增结果如图2.图2-B 中1一7分别表示50〜62 C条件，每隔2度设置一个梯度值，在该条件下复性，持续35个循环.图2-B 中50 C温度条件菌液能看出很明显很亮的单一条 带，52,54,56,58,60,62 C温度条件菌液均无条带 (表2),可知黄色蛋白质粒P C R最适温度为50 C.以绿色质粒G F P104过夜单菌落摇床菌液为模板，使用高保真酶进行P C R,扩增结果如图2.图2-C中1一8中分别代表50〜64 C条件，每隔2个温度单位为一个梯度，在该条件下进行复性，持续35个循环，图2-C中的50,52,54,56,58,60,62,64 C 温度条件下均可以清楚的看到很亮的单一条带（表2),可知绿色蛋白质粒P C R适宜温度为50〜64 C.2 3红、黄、绿3种不同颜色蛋白质粒的碱基序列随机挑选热激转导后保存至30%甘油管，重复3次测序比对，测序结果理想，同源序列完全相同，其菌液均扩出大小一致的片段，均获得了大小接近 780 b p的产物.红色质粒R e d l23蛋白重复3次测序结果与基因库比对匹配率为98%,表明红色质粒 R e d l23成功转导至大肠杆菌.黄色质粒Y F P69蛋白重复3次测序结果与基因库比对匹配率为96%, 表明黄色质粒Y F P69成功转导至大肠杆菌.绿色质粒G F P104蛋白重复3次测序结果与基因库比对匹配率为98%,表明绿色质粒G F P104成功转导至大 肠杆菌．2.4红、黄、绿3种不同颜色蛋白质粒转导的稳 定性3种不同颜色蛋白质粒均用荧光显微镜压片单 菌落，观察有无荧光，均能明显的看到红色、黄色和绿色的荧光(表3)，如图3所示，表明红、黄、绿3种不同颜色蛋白质粒已被成功导入大肠杆菌中，并均能稳定表达.表2不同温度下红、黄、绿3种蛋白质粒PCR鉴定Table2Identification of three kinds of plasmid protein PCR of redyellow and green at different temperatures颜色不同温度菌液PCR鉴定情况50C52C54C56C58C60C62C64C红色质粒十十十一一一一黄色质粒十一一一一一一绿色质粒十十十十十十十十+指PCR鉴定清楚看见明显很亮的单一条带，一表示PCR鉴定不能看见明显很亮的单一条带.第3期杨晓玫等:红、黄、绿三种颜色荧光质粒导入大肠杆菌中的稳定性表达197 M:1000Marker;l:Redl23、YFP69、GFP104PCR产物；1—7分别对应50,52,54,56,58,60,62 €下的扩增产物.图2Redl23、YFP69、GFP104菌液PCR鉴定Figure2R edl23^YFP69^G FP l04Electrophoreis of colony PCRRFP YFP GFP图3红、黄、绿3种荧光蛋白显微镜下观察的不同颜色荧光Figure3Red^yellow and green three different color fluorescent protein fluorescence microscope表3红、黄、绿3种蛋白质粒荧光显微镜下不同荧光情况Table3Fluorescence spectra of three plasmidproteins of red,yellow and green under differentfluorescence conditions颜色红色蛋白黄色蛋白绿色蛋白质粒质粒质粒荧光显微镜观+++察荧光情况十指荧光显微镜下可以看到明显的有颜色荧光，一表示荧光显微镜下不能看到明显的有颜色荧光.3讨论国内对荧光蛋白质粒的研究已取得多项成 果[11]，但是基于对大肠杆菌基因表达的研究，以荧光质粒作为转导基因来研究辅助菌大肠杆菌的转导报道很少.本试验通过划分不同的浓度和温度，研究不同颜色荧光蛋白质粒转导大肠杆菌的条件，确定稳定性好的携带荧光蛋白质粒的大肠杆菌.转导大肠杆菌目前有热激转导和电击转导两种方法，但是目前关于荧光蛋白质粒转导大肠杆菌的 研究还相当匮乏，对比韩颖等[12]、李晶等[13]、孙彦等[14]研究的热激转导方法条件与宋诗铎等[15]研究的电击转导方法条件，本试验通过大肠杆菌D H5a 热激法转导，与王萍等[6]研究表明的大肠杆菌转导 最适温度和大肠杆菌感受态细胞最适保存温度一 致，已将外源红黄绿荧光质粒D N A成功的转化至 大肠杆菌中，大肠杆菌异源表达高分子量型性质与 能力[17]，传代稳定，外源基因在其中的表达产物热 稳定性不变.试验结果表明不同颜色荧光质粒D N A 均能进行异源转导，能按照规定成功转化至大肠杆 菌，作后续三亲本杂交试验的供体菌株，进行对苜蓿植株荧光标记，更加直观简便观察根瘤菌移动情况，能提高植物的产量[18].荧光报告质粒的转化，为进198甘肃农业大学学报2018 年一步研究荧光基因的功能和表达提供了一个有效的前提.目前，本研究组正利用青色荧光基因检测苜蓿里的根瘤情况，颜色种类越多越具有多元化，对比较分析各种颜色在苜蓿里的生存差异及最适部位，具有重要研究意义.对荧光蛋白蛋白质粒产生的荧光蛋白自主发光作用已经做过广泛的研究.郭永明等[19]研究表明使 用荧光显微镜对荧光的转导结果可以进行直观的观察，本试验研究结果均已用显微镜可以直观清晰的 看到3种荧光的特定颜色.鉴于实践以及理论研究 的目的，现在人们的兴趣更多集中在荧光蛋白示踪 作用、发光机制、重组D N A的结构与功能的关系，以及其他科学研究应用等问题上.迄今为止，只有一种青色荧光蛋白(绿色荧光的变异)和大肠杆菌通过三亲本杂交成功结合，在转导荧光质粒的研究中发现，红、黄、绿3种颜色和大肠 杆菌结合，条件略有不同，黄色对温度要求较高，黄色和绿色在原质粒D N A稀释10倍的条件下转化，红色直接可以对原质粒D N A进行转化.正常条件 下质粒通常会保存到大肠杆菌里，直接进行三亲本 杂交试验，基于试验材料是张学贤博士惠赠的质粒 D N A,要成功的进行三亲本杂交试验，质粒D N A不能直接进行杂交试验，必须要把质粒借助于大肠杆 菌才可以进行杂交，本试验成功的运用D H5a法将荧光质粒D N A转导到大肠杆菌中，将为整个荧光 蛋白质粒转化提供一定的方法学基础，为后续杂交 试验奠定了基础，具有重要的研究意义.参考文献[1]田涛,亓雪晨，王琦，等.芽孢杆菌绿色荧光蛋白标记及其在小麦体表定殖的初探[〗].植物病理学报,2004,4(4)346-351.[2] M A T Z M V,F R A D K O V A F,L A B A S Y A ,et al.Fluorescent proteins from nonbiolum nescent Antho-zoa species[]].N at Biotechnol,1999,17(10) ：969-973.[3] M?S P,BE A C H Y R N.Role of microtubules in the intracellular distribution of tobacco mosaic virus movement protein[J],Proc N atl Acad Srt U S A,2000, 97(22)12345-12349.[4] JA C H G,B IN O T E,S A B IN E F,e t e of red flu orescent protein from Discosomasp. (dsRED)as a reporter fo r plant gene expression[J],Plant J,2001,28(4)：483-491.[5]孙金钰，郑屹峰,巫启明，等，环状排列黄色荧光蛋白在大肠杆菌及毕赤酵母中表达的比较研究[].福州大学学报（自然科学版），2014,42(5)795800.[6]原荣荣，夏远，等.大肠杆菌信号转导系统研究进展[].绿色科技,2013,6(6):310-313.[7]周俊初,使小娟，谢波.绿色荧光蛋白基因（gfp)在华癸中生根瘤菌与紫云英共生固氮作用研究中应用[J].中国科学基金,2001,5(19):4851.[]乐易林.大肠杆菌热激反应研究及其在重组蛋白表达 中的应用[J].微生物学通报，2013,40(11):20902096[9]冯光燕，王学敏,付媛媛，等.紫花苜蓿M sW RKY33转录因子的分离及遗传转化研究[J].草业学报,2015,24(11)49-57.[10]马玲珑.大麦成熟胚离体培养条件的优化及H gN-H X1基因表达载体的构建和遗传转化[D].兰州：甘肃农业大学,015.[11]杨杰、张智红、胳清铭.荧光蛋白研究进展[J].生物物理学报，2010,26(11)1025-1035.[12]韩颖,赵寿经，杨瑜，等.玉米陈化关键酶基因Zmlox-2的R N A干扰载体的构建[J].华南理工大学学报(自然科学版），2016,3(44)136-140.[13]李晶，任卫波,郭慧琴，等.农杆菌介导的狋富硒基因转化紫花苜蓿研究[J].草业科学，2012, 29(8):1224-1228[14]孙彦，郭校民，周禾，等.白颖苔草热激转录因子(HSF1)真核表达载体的构建[J].草业科学,2012,29(4）544-548[15]宋诗铎,张同海，祁伟，等.应用电激法在大肠杆菌中导人外源性D N A[J].生物工程学报,1993,9(3):237-240[16]王萍,殷春燕，盈磊.不同方法转化大肠杆菌和农杆菌转化效率的研究[].淮海工学院学报（自然科学版），2007,16(2)56-58.[17]张晓欢，崔文璟,周哲敏，等.高分子量腈水合酶在大肠杆菌中的表达策略及重组菌的细胞催化[].植物学通报，2016,4(30)2121-2128.[18]陈力玉，张淑卿,李剑锋，等.接种荧光标记根瘤菌对苜蓿幼苗生长的影响[J].草原与草坪,2013,33(6)18. [19]郭永明,陈晓丽，余晶，等.致病性大肠杆菌绿色荧光蛋白标记[J].川北医学院学报,2015,30(5)583-585.(责任编辑李辛）。
英语国际会议PPT课件
Materials and Methods
Patients
Materials and Methods
Cytokine assessment by ELISA
Western blot
Cell isolation and culture
Statistical analysis
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Methods
IFN-c-induced protein of 10-kDa (IP-10)/CXCL10
we studied the effects of α-toxin on Th1- and Th2related chemokines in macrophages from patients with AD and psoriasis where the intrinsic abnormal and different chemokines production profile is well defined.
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Figure 3 Punch biopsies (3 mm) from healthy individuals were left either unstimulated (A) or stimulated with a-toxin (100 ng/ ml) (B) or IFN-c (100 ng/ml) (C) for 24 h at 37C. 5-lm paraffin sections were stained for CXCL10 along with appropriate isotype as well as CD68.
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Low effect of a-toxin on CXCL10 induction (Th1-related chemokine) in macrophages from patients with AD
Progress in the expression of P-glycoprotein in br
ReviewProgress in the expression of P-glycoprotein in brain metastases of lung cancer andrelated TCM researchShuai Zhui，#, Ling_Yu Qii，#, Xue Wang2，#，Hua_Yao Li、Jia Li、Jing Yang3*1Shandong University of TCM, Jinan 250000, China. 2Qingdao University, Qingdao 266071, China. 3Department of Oncology，Weifang Traditional Chinese Hospital，Weifang 261031，Shandong Province，China.# These authors contributed equally to this work.*Correspondence to: Jing Yang, Department of Oncology, Weifang Traditional Chinese Hospital, Weifang 261031，Shandong Province, China. E-mail: zhongliuyike@.AbstractBrain metastases are common intracranial tumors, and their occurrence not only represents a high degree of malignancy, but also often is the major factor in treatment failure and poor prognosis. Primary site of brain metastases often occur in lung. P_glycoprotein is a member of the (ATP binding cassette) transporter superfamily，which is closely related to the development of lung metastases. It is the main reason for influencing the drug through the blood_brain barrier into the brain tissue, and it also is an important factor affecting the treatment of brain metastases. According to the theory of traditional chinese mddicine, the pathogenesis of brain metastases is due to phlegm, poison, stasis, virtual and so on. The principle of treatment is to promote blood circulation, remove phlegm turbidity. In recent years, the impact of Chinese herbal medicine on P_glycoprotein is increasing. This paper analyzes the mechanism and components of the relevant Chinese medicine on P_glycoprotein. It provides a reference for clinical rational drug use.Keywords:Lung cancer; Brain metastasis; P-glycoprotein; Blood-brain barrier; Chinese medicine treatment; Research progress摘要脑转移瘤是常见的颅内肿瘤，其发生不仅代表恶性程度高，往往也是治疗失败、预后差的主要因素。
小学下册第1次英语第四单元测验卷(有答案)
小学下册英语第四单元测验卷(有答案)英语试题一、综合题(本题有100小题，每小题1分，共100分.每小题不选、错误，均不给分)1.We have a ______ of books in the library. (collection)2.The main product of cellular respiration is ______.3.The _____ (小鸭) follows its mother everywhere.4.My dad is my strong _______ who always protects me.5. A __________ is formed when minerals crystallize from cooling magma.6.The owl can turn its head almost ______ (完全).7.What is the capital city of Egypt?A. CairoB. LuxorC. AlexandriaD. Giza答案:A8.What is the term for a baby chicken?A. DucklingB. ChickC. GoslingD. Calf答案:B Chick9.My parents are very _______ (支持我的).10.My best friend's family has a _______ (动物). 它的性格很 _______ (形容词).11.The _____ (钱包) is on the table.12. A sunny day makes me feel very ______ (有活力).13.What is the name of the phenomenon where the moon blocks the sun?A. Solar eclipseB. Lunar eclipseC. SupermoonD. Blood moon答案:A14.Hubble's Law relates to the expansion of the ______.15.I enjoy taking ______ (旅行) to different countries to learn about their ______ (文化).16.My sister enjoys __________ (游览博物馆).17.Which season comes after spring?A. WinterB. SummerC. FallD. Autumn答案:B18.My _____ (玩具赛车) is very fast.19.The _____ (科技) helps us understand plants better.20.I can ___ (jump) over the puddles.21.What is the name of the famous statue in Rio de Janeiro?A. Christ the RedeemerB. Statue of LibertyC. DavidD. Venus de Milo答案:A22.This ________ (玩具) is perfect for group activities.23.What is the name of the device used to measure temperature?A. BarometerB. ThermometerC. HydrometerD. Anemometer答案:B24.The __________ (历史的探索) unveils insights.25. A _____ (植物实验) can teach students about biology.26.What is the name of the famous painting by Leonardo da Vinci?A. The Starry NightB. The Mona LisaC. The Last SupperD. The Scream27.The ancient Greeks believed in the importance of ______ (教育).28.My aunt loves to do ____ (photography).29.I like to make ___ (friends) everywhere I go.30.The baby is ________ in the crib.31.The _____ is the temperature of space.32.My uncle loves to __________ (钓鱼) on weekends.33. A ________ (北极熊) lives in the cold Arctic and is very large.34.We have a _____ (庆祝) for the holiday.35.What do we call the study of human behavior and mental processes?A. PsychologyB. SociologyC. AnthropologyD. Geography答案:A36.The __________ is home to many unique species of animals.37.The car is parked ________ the garage.38.我最喜欢的老师是 _______ (名字). 她的教学风格很 _______ (形容词).39.The chemical symbol for manganese is ______.40. A ____ is a small mammal that loves to dig.41.What do we call the person who studies stars?A. BiologistB. AstronomerC. GeologistD. Chemist42.I have a ______ that I take care of.43.What is the name of the first artificial satellite launched into orbit?A. Sputnik 1B. Explorer 1C. Vanguard 1D. Luna 144.The __________ (环保活动) raise awareness of issues.45.What do we call the act of making a plan?A. OrganizingB. PlanningC. StrategizingD. Arranging46.What is the name of the famous American author known for his adventure novels?A. Mark TwainB. Ernest HemingwayC. F. Scott FitzgeraldD. John Steinbeck答案:A47.What do we call a baby chicken?A. KittenB. PuppyC. ChickD. Calf答案:C48.Every Christmas, I hope for a new ____. (玩具名称)49.How many teeth does an adult human have?A. 28B. 30C. 32D. 34答案:C50. A compound can have unique ______.51.What is the boiling point of water in Celsius?A. 50B. 100C. 0D. 20052.What do we call a person who studies the effects of drugs on the body?A. PharmacologistB. ToxicologistC. BiochemistD. Chemist答案:A53.The _______ (鱼) swims gracefully in the water.54.What do we call a young alligator?A. HatchlingB. PupC. KitD. Chick答案:A Hatchling55.The __________ can be quite unpredictable in spring. (天气)56.An unstable nucleus can undergo _____ (radioactive decay).57.The playground is full of ______.58. A __________ is a large body of saltwater.59. A precipitate forms when two liquids ______.60.The ancient Egyptians made ______ (珠宝) from gold and gems.61.The _____ (soup) is hot.62.I see a ______ in the sky. (cloud)63.What is the color of snow?A. WhiteB. BlackC. GrayD. Blue64.______ can be a sign of a healthy ecosystem. (植物的多样性可以是健康生态系统的标志。
G蛋白偶联雌激素受体对乳腺癌患者预后和临床病理的影响：系统荟萃分析
doi:10.3971/j.issn.1000-8578.2023.23.0237·临床研究·G蛋白偶联雌激素受体对乳腺癌患者预后和临床病理的影响：系统荟萃分析刘龙娇，姚宇锋Effect of G-protein-coupled Estrogen Receptor on Prognosis and Clinicopathological Characteristics of Patients with Breast Cancer: A Systematic Meta-analysisLIU Longjiao, YAO YufengDepartment of General Surgery, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nanjing Medical University Affiliated Cancer Hospital, Nanjing 210000, China CorrespondingAuthor:YAOYufeng,Email:**********************Abstract: Objective In this study, a meta-analysis of the expression of G-protein-coupled estrogen receptor (GPER) in breast cancer (BC) and its role in prognosis was conducted to understand the effect of this expression on the survival and clinicopathological characteristics of patients with BC. Methods Identical search strategies were used to search relevant literature in electronic databases updated to November 24, 2022. Individual hazard ratios (HR s) and odds ratios (OR s) with their 95%CI were extracted and pooled to evaluate the strength of the association between positive GPER expression and survival results, the clinicopathological features of patients with BC. Begg’s tests, Egger’s tests, and funnel plots were used to evaluate publication bias. Heterogeneity and sensitivity were also assessed. All works were completed using Review Manager 5.4.1. Results GPER expression had a favorable effect on OS (HR=0.77; 95%CI: 0.49-1.22; Z=1.10; P=0.27) and an unfavorable effect on DFS/RFS/DDFS (HR=1.03; 95%CI: 0.64-1.65; Z=0.13; P=0.90) in patients with BC. GPER expression was not significantly related to the prognosis of patients with BC, and GPER expression was not an independent prognostic factor. Furthermore, GPER expression was significantly associated with TNM staging (OR=0.31, 95%CI: 0.06-0.55, Z=2.43, P=0.02), distant metastasis (OR=6.82, 95%CI: 1.89-24.55, Z=2.94, P=0.003), histological grade (OR=0.009, 95%CI: −0.16-0.01, Z=2.16, P=0.03), ER expression (OR=1.77, 95%CI: 1.15-2.72, Z=2.59, P=0.009), and PR expression (OR=1.36, 95%CI: 1.00-1.84, Z=1.95, P=0.05). Conclusion GPER may not be an independent prognostic factor for BC. GPER expression was significantly related to some clinicopathological features of patients with BC, including TNM staging, distant metastasis, histological grade, ER expression, and PR expression.Key words: GPER; Breast cancer; System meta-analysis; Prognosis; Clinicopathological characteristicsCompeting interests: The authors declare that they have no competing interests.摘 要：目的 本研究对G蛋白偶联雌激素受体（GPER）在乳腺癌中的表达及其对预后的作用进行荟萃分析，以了解GPER表达对乳腺癌患者生存和临床病理特征的影响。
甲型副伤寒沙门氏菌HmpA蛋白的表达纯化及生物学特征预测
王磊，刘小草，董雨，等. 甲型副伤寒沙门氏菌HmpA 蛋白的表达纯化及生物学特征预测[J]. 食品工业科技，2023，44（24）：131−138. doi: 10.13386/j.issn1002-0306.2023010136WANG Lei, LIU Xiaocao, DONG Yu, et al. Expression, Purification and Biological Characteristics Prediction of Protein HmpA of Salmonella paratyphi A[J]. Science and Technology of Food Industry, 2023, 44(24): 131−138. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2023010136· 生物工程 ·甲型副伤寒沙门氏菌HmpA 蛋白的表达纯化及生物学特征预测王 磊，刘小草，董 雨，王雪婷，柴徵微，李 吉，丁爱军，张维明，曾韦锟*（昆明学院医学院，云南昆明 650214）摘 要：目的：原核表达甲型副伤寒沙门氏菌（Salmonella paratyphi A ）HmpA 蛋白，并对其进行生物信息学分析，为研究副甲菌HmpA 蛋白对于宿主体内一氧化氮（NO ）信号通路的影响提供理论参考。
方法：PCR 扩增hmpA 基因，亚克隆至T 载体后，再构建pNdeI-hmpA 表达载体，转化BL21（DE3），经IPTG 诱导后，SDS-PAGE 检测蛋白表达形式，利用Histrap 预装柱亲和纯化HmpA 蛋白，并用Western blot 鉴定，生物信息学多方法分析HmpA 蛋白特征。
结果：成功构建了原核表达载体pNdeI-hmpA ，低温诱导时，HmpA 蛋白以包涵体和可溶表达形式并存；纯化后的HmpA 蛋白可被His 标签抗体检测。