Chapter six Molecular Cloning

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Chapter 4 Molecular Clonning Methods

Chapter 4 Molecular Cloning Methods第四章分子克隆方法4.1 基因克隆分子克隆概述•分子克隆（molecular cloning，基因克隆）：通过体外重组技术，将一段目的DNA经切割、连接插入适当载体，并导入受体细胞，扩增形成大量子代分子的过程•基因克隆的核心---体外重组（Recombination）:人工将一段目的DNA插入一个载体的过程。

孙悟空也会“克隆”一个细菌经过20分钟左右就可一分为二；仙人掌切成几块，每块落地就生根，......凡此种种，都是生物靠自身的一分为二或自身的一小部分的扩大来繁衍后代，这就是无性繁殖。

无性繁殖的英文名称叫“Clone”，音译为“克隆”。

实际上，英文的“Clone”来源于希腊文“Klone”，原意是用“嫩枝”或“插条”繁殖。

时至今日，“克隆”的含义已不仅仅是“无性繁殖”，凡来自一个祖先，无性繁殖出的一群个体，也叫“克隆”。

可以这样说，关于克隆的设想，我国明代的大作家吴承恩已有精彩的描述--孙悟空经常在紧要关头拔一把猴毛变出一大群猴子，就是克隆猴。

4.1.1 限制性内切酶什么是限制性内切酶？限制性核酸内切酶（restriction endonuclease）：识别并切割特异的双链DNA序列的一种内切核酸酶。

[别名]Endodeoxyribonuclease[单位定义]在适当的温度下，在1小时内，1μgDNA在50μl体积中，被完全消化所需的限制性内切酶的量。

一般说来，线性DNA比超螺旋DNA更易消化。

限制性内切酶的发现•19世纪60年代末期，Stewart Linn和Werner Arbert从E.coli中发现了限制性核酸内切酶。

这些酶通过切断外源DNA来防止外源DNA，如病毒DNA的入侵，可以说它们“限制”了病毒的宿主范围，且它们的切割位点在外源DNA的内部，因此称限制性核酸内切酶。

•1971 美国人Daniel Nathans 和Hamilton Smith 发展了核酸酶切技术。

分子克隆

3.与反转录相关的PCR扩增
RT-PCR（reverse transcriptase-PCR,RT-PCR): 又称反转录PCR, 是以反转录的cDNA作模板所进行的PCR，
可对基因的表达和序列多态性分析。
ＲＴ－ＰＣＲ
反转录
逆转录酶
AAAnA mRNA
AAAnA
T T TnT
cDNA第一链
ＤＮＡ聚合酶 cDNA
目的基因的获取-----PCR技术:
定义： PCR技术又称聚合酶链式反应（polymerase chain reaction)，是通过模拟体内 DNA 复制的方式，在体外选择性地将 DNA 某个特殊区域扩增出来的技术。
Taq DNA多聚酶的发现
Heat-stable polymerase is vital to the ease of the process…
4.PCR反应程序
⑴94~96℃ ⑵94℃ ⑸ 25- ⑶50-60℃ 35个循 ⑷72℃ 环 ⑹72℃ ⑺4-10℃ 30’’-3’ 预变性（使模板DNA充分变性） 30’’ 变性 30’’-1’ 复性（使引物与模板充分退火） n’(按1’扩增1kb计算）延伸 3-7’ 总的延伸（使产物延伸完整）保存
质粒自身含有复制起始点，与相应的顺式调控元件组成一个复制子(replicon)，能利用细菌的酶系统进行独立的复制及转录。质粒具有多种遗传选择标记，包括各种抗药基因或营养代谢基因等。
氨苄青霉素抗性(ampicillin resistance，ampr)基因：
此基因编码ß 内酰胺酶，该酶能水解氨苄青霉素ß —内酰胺环，使之失效而使细菌产生耐药。
1988年Saiki 等从温泉（ Hot spring）中分离的一株水生嗜热杆菌 (thermus aquaticus) 中提取到一种耐热DNA 聚合酶。

Chapter 6 Cloning Vectors for E and eukaryotes

Chapter 6 Cloning Vectors for E.coli and eukaryotes
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Contents

6.1 Cloning vectors based on E.coli plasmids 6.1.1 The nomenclature of plasmid cloning vectors 6.1.2 The useful properties of pBR322 6.1.3 Other typical E.coli plasmid cloning vectors 6.1.5 pMD-19T 6.2 Vectors for yeast and other fungi 6.2.1 Vectors based on the 2 m plasmid 6.2.2 A Yep may insert into yeast chromosomal DNA 6.2.3 Other types of yeast cloning vector 6.2.4 Artificial chromosomes can be used to clone large pieces of DNA in yeast 6.2.5 Vectors for other yeasts and fungi
A map of pBR322
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6.1.3 The pedigree of pBR322
Fig 6.2 The pedigree of pBR322:(a) the manipulations involved in construction of pBR322, and (b) a summary of the origins of pBR322
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contents

氯化锂沉淀rna原理

氯化锂沉淀rna原理一、引言RNA（核糖核酸）是生物体内一类重要的核酸分子，具有传递遗传信息、调控基因表达等重要功能。

为了研究RNA的结构和功能，科学家们开展了许多RNA的提取和纯化研究。

其中，氯化锂沉淀RNA是一种常用的方法之一。

二、氯化锂沉淀RNA的原理氯化锂沉淀RNA的原理是利用氯化锂与RNA中的磷酸根结合形成沉淀，从而将RNA分离出来。

具体步骤如下：1. 细胞破碎：将待提取的RNA的细胞破碎，使RNA释放到溶液中。

2. 细胞裂解液处理：加入细胞裂解液，使细胞内的蛋白质、DNA等杂质被裂解。

3. 加入氯化锂溶液：加入氯化锂溶液，使细胞裂解液中的RNA与氯化锂结合形成沉淀。

4. 沉淀处理：通过离心将沉淀与上清液分离开来。

5. 沉淀洗涤：利用乙醇洗涤沉淀，去除杂质。

6. RNA溶解：将沉淀中的RNA用适当的溶液进行溶解，得到纯化的RNA溶液。

三、实验操作1. 提取RNA的样品准备：准备待提取RNA的样品，可以是细菌、真核生物细胞等。

2. 细胞破碎：使用细胞破碎液将细胞破碎，使细胞内的RNA释放到溶液中。

3. 细胞裂解液处理：加入细胞裂解液，使细胞内的蛋白质、DNA等杂质被裂解。

4. 加入氯化锂溶液：根据实验所需的RNA浓度，加入适量的氯化锂溶液，使RNA与氯化锂结合形成沉淀。

5. 沉淀处理：将混合物进行离心，使沉淀与上清液分离开来。

6. 沉淀洗涤：用75%乙醇洗涤沉淀，去除杂质。

7. RNA溶解：用适当的溶液（如RNase-free水）将沉淀中的RNA 溶解，得到纯化的RNA溶液。

四、注意事项1. 实验操作时应注意严格遵守无菌操作规范，以保证RNA的纯度和完整性。

2. 实验过程中应避免RNase的污染，如需使用工具、试剂等，应先进行RNase去污处理。

3. 实验室中的仪器和试剂应保持干净，以避免杂质对RNA提取的干扰。

4. 操作过程中应注意个人安全，如需使用有毒试剂，应戴好防护手套和口罩。

五、总结氯化锂沉淀RNA是一种常用的RNA提取和纯化方法，其原理是利用氯化锂与RNA中的磷酸根结合形成沉淀。

分子克隆英文版

分子克隆英文版Molecular CloningMolecular cloning is a fundamental technique in modern molecular biology and biotechnology, allowing for the isolation, amplification, and manipulation of specific DNA sequences. This process involves the transfer of a DNA fragment from one organism to a self-replicating genetic element, such as a plasmid or a virus, which can then be introduced into a host cell. The host cell, typically a bacterium or a eukaryotic cell, then replicates the inserted DNA, producing multiple copies of the desired genetic material.The primary motivation behind molecular cloning is the need to obtain large quantities of a specific DNA sequence for various applications, including genetic research, disease diagnosis, and the production of recombinant proteins. By cloning a gene of interest, researchers can generate an unlimited supply of the target DNA, enabling further analysis, modification, and utilization.The process of molecular cloning can be divided into several key steps. The first step involves the isolation of the desired DNA fragment, which can be obtained from a variety of sources, such asgenomic DNA, complementary DNA (cDNA), or synthetic DNA. This DNA fragment is then inserted into a suitable vector, such as a plasmid or a viral genome, using specialized enzymes called restriction endonucleases and DNA ligase.The resulting recombinant DNA molecule is then introduced into a host cell, typically a bacterial cell like Escherichia coli, through a process called transformation. The transformed host cells are then cultured, and the cells containing the desired recombinant DNA are selected and amplified. This amplification process allows for the production of large quantities of the target DNA sequence.One of the most important applications of molecular cloning is the production of recombinant proteins. By cloning a gene encoding a specific protein, researchers can express and purify the protein in a host cell, such as a bacterium or a eukaryotic cell line. This technique has been instrumental in the development of numerous therapeutic proteins, including insulin, growth hormones, and monoclonal antibodies, which have revolutionized the field of medicine.Molecular cloning is also essential for the study of gene function and regulation. By cloning a gene and introducing it into a host cell, researchers can investigate the expression, localization, and interactions of the encoded protein, as well as the regulatory mechanisms that control its activity. This knowledge is crucial forunderstanding the complex biological processes that underlie human health and disease.In addition to its applications in research, molecular cloning has also found widespread use in the field of genetic engineering. By modifying the genetic material of organisms, scientists can create genetically modified organisms (GMOs) with desirable traits, such as increased crop yield, improved nutritional value, or resistance to pests and diseases. This technology has significant implications for agriculture, environmental conservation, and the development of new therapeutic strategies.Despite its many benefits, molecular cloning is not without its challenges and ethical considerations. The potential for misuse or unintended consequences of genetic manipulation has sparked ongoing debates and the need for robust regulatory frameworks to ensure the responsible and ethical use of this powerful technology.In conclusion, molecular cloning is a fundamental technique in modern molecular biology and biotechnology, enabling the isolation, amplification, and manipulation of specific DNA sequences. This technology has revolutionized numerous fields, from genetic research and medicine to agriculture and environmental conservation. As the field of molecular biology continues to evolve, the applications and implications of molecular cloning willundoubtedly continue to expand, posing both exciting opportunities and important ethical considerations for the future.。

Molecular Cloning and Characterization

Molecular Cloning and Characterization of a Guanylyl Cyclase,PnGC-1,Involved in Light Signaling in Pharbitis nilAdriana Szmidt-Jaworska ÆKrzysztof Jaworski ÆAgnieszka Pawełek ÆJan KopcewiczReceived:20April 2009/Accepted:12May 2009/Published online:22June 2009ÓSpringer Science+Business Media,LLC 2009Abstract Guanylyl cyclases (GCs)are enzymes involved in the biosynthesis of cyclic guanosine monophosphate (cGMP).Here we report the cloning and characterization of a new guanylyl cyclase,designated PnGC-1,from Phar-bitis nil .This gene encodes a protein of 286amino acids,with a calculated molecular mass of 32kDa.The predicted amino acid sequence contains all typical features and shows high identity with known plant GCs.The GST-PnGC-1was catalytically active in E.coli cells and the puriﬁed,recombinant PnGC-1was able to convert GTP to cGMP in the presence of Mn 2?.Moreover,the enzyme activity was strongly inhibited by a speciﬁc sGC inhibitor,NS2028,whereas in the case of nitric oxide,an animal sGC stimulator,no positive effect was observed.Besides the identiﬁcation of the PnGC-1as a guanylyl cyclase,it was shown that a transcript of PnGC-1was present in every tested organ of the light-or dark-grown plants;however,the highest expression level was found in dark-treated plants.The PnGC-1mRNA level in the cotyledons exhibited diurnal oscillations under short-day conditions (8/16-h photoperiod).Meanwhile,monitoring of transcript levels in cotyledons exposed to a special photoperiodic regime (24h light of low intensity then 24h long night with or without far-red light before the night)revealed that a stabile phytochrome is involved in this process.These data unequivocally identify the product of the PnGC-1gene as a guanylyl cyclase and emphasize the potential that soluble GC can be an element of light signal transduction.Keywords Guanylyl cyclase ÁcGMP ÁLight signaling ÁPharbitis nilIntroductionThe intracellular messenger 30,50-cyclic guanosine mono-phosphate (cGMP)plays an important role both in animal and plant cells.The enzymes responsible for cGMP syn-thesis are guanylyl cyclases that are a set of cytosolic and membrane proteins (Murad 1994;Lucas and others 2002).Guanylyl cyclase catalyzes the conversion of GTP to cGMP in response to various extra-and intracellular stimuli,thereby providing an important second messenger for the regulation of protein kinases,phosphodiesterases,and ion channels (Newton and others 1999).Although cGMP is supposed to be a putative interme-diary involved in signal transduction,its speciﬁc position in signal transduction and the mechanism of its action in plant cells remains unknown.Since the discovery of cGMP in plant cells,it has become clear that an enzyme responsible for its synthesis must also be present (Newton and Smith 2004).However,in plants,the knowledge about the enzymes involved in cGMP metabolism is insufﬁcient.Only a few studies on plant GC have been conducted.For example,in intact chloroplasts from Phaseolus vulgaris the speciﬁc GC activity was analyzed using the conversion of labeled [32P]-GTP into [32P]-cGMP (Newton and others 1984).Volotovsky and others (2003)revealed the activity of guanylyl cyclase in plasma membranes in Avena sativa cells.In Pharbitis nil an enzyme with GC activity was localized in cytoplasm and its activity changed in various light conditions (Szmidt-Jaworska and others 2008b ,2009).A.Szmidt-Jaworska (&)ÁK.Jaworski ÁA.Pawełek ÁJ.KopcewiczDepartment of Physiology and Molecular Biology of Plants,Institute of General and Molecular Biology,NicolausCopernicus University,Gagarina St.9,87-100Torun,Poland e-mail:asjawors@umk.plJ Plant Growth Regul (2009)28:367–380DOI 10.1007/s00344-009-9105-8Recently,important advances using molecular and genetic approaches have been made in plant GC research.A small gene family of GCs encoding different isoforms of the enzyme has been identiﬁed.There are three reports of cloning of putative cyclase from higher plants(Ludidi and Gehring2003;Kwezi and others2007;Yuan and others 2008).A motif search of the Arabidopsis genome based on conserved and functionally assigned amino acids in the catalytic domain of GCs returned one candidate(AtGC-1) that contained the glycine-rich motif typical for GCs. When AtGC-1was expressed in E.coli,the cell extract contained2.5times more cGMP than the control one.The amino acid sequence of protein indicates that it is soluble-type cyclase,unaffected by NO(Ludidi and Gehring2003). Using the same strategy,Kwezi and others(2007)cloned and expressed a recombinant protein(AtBRI1-GC).This brassinosteroid receptor harbors the putative catalytic domain and can convert GTP to cGMP in vitro.It seems that AtBRI1-GC may belong to a novel class of cyclases that contains a GC and cytosolic kinase domain.Recently, a guanylyl cyclase-like gene from Zea mays has been cloned and characterized(Yuan and others2008).Multiple copies of this gene have been mapped and it was shown that gene expression is associated with Fusarium grami-nearum resistance.Cyclic GMP as a second messenger plays an essential role in many important cellular processes.In animal and plant cells,transient changes in cGMP level in the cytosol have been observed during growth,development,and under stress conditions(Cousson2001;Maathuis2006). Signiﬁcant changes in cGMP levels have been reported in response to light treatment,in phytochrome-dependent gene expression required for chloroplast development and anthocyanin biosynthesis(Bowler and others1994),gib-berellic acid-dependent a-amylase synthesis(Penson and others1996),and photoperiodicﬂower induction(Szmidt-Jaworska and others2004,2008a).It is known that the endogenous level of cGMP can be modulated by either controlling the activity of GCs,of cyclic nucleotide phosphodiesterase,or of both.Moreover, cGMP formation may be regulated also indirectly at the transcriptional and post-transcriptional levels(Jiang and Stojilkovic2006).The mechanisms by which plant GC can be regulated are important questions which touch upon molecular cloning of the GC gene as well as analysis of the expression of GC coding gene(s),especially since expres-sion of the plant GC was not tested by any of the constructs reported in the earlier works.Studies focusing on such a problem and identiﬁcation of the GC gene in P.nil are the subject of this report.Moreover,the biochemical charac-terization of the recombinant protein is described and conﬁrms that the analyzed enzyme belongs to a class of plant GCs.Materials and MethodsPlant Material and Light TreatmentsThe investigations were conducted on5-day-old seedlings of morning glory(Pharbitis nil L.Chois),the Japanese variety Violet(Marutane Seed Co.,Kyoto,Japan).Seeds were soaked in concentrated sulfuric acid for50min and then washed in running tap water for3h.They were left in distilled water at25°C overnight.The swollen seeds were planted on a mixture of vermiculite and sand(2:1w/w)and grown at25°C in various light conditions.For expression analysis in vegetative organs,plants were grown in darkness or under continuous light(130l mol m-2s-1,white lightﬂuorescent tubes,Polam)for5days. The cotyledons,hypocotyls,and roots were picked,imme-diately frozen in liquid nitrogen,and stored at-80°C.For expression analysis in various light/dark regimes in variant I,plants were growing in continuous light for5days.A portion of5-day-old plants was left in such conditions. The rest of the plants were exposed to a16-h-long darkness, but for some of them the long night was disrupted by a 5-min-long pulse of red light(R,1.5l mol m-2s-1,ﬂuo-rescent tubes TLD15R/18W,Philips)at the8th hour or R followed by a10-min-long pulse of far-red light(FR, 0.1l mol m-2s-1,narrow bandﬁlter FR=730±2,half-bandwidth=9nm).In variant II,plants were grown for 4days in darkness and then cultivated for24h in low-intensity white light(40l mol m-2s-1,cool whiteﬂuo-rescent tubes,Philips).A portion of these plants was left in such conditions.The rest were moved to darkness for24h (long inductive night).Some of the seedlings were irradiated with a10-min-long pulse of FR light at the end of the24-h white-light period or FR light followed by R light.Afterward,for both variants,cotyledons were harvested every hour,immediately frozen in liquid nitrogen,and stored at-80°C.Molecular Cloning of PnGC-1Molecular cloning of PnGC-1consisted of three steps: PnGC-1fragment production by PCR,30RACE(rapid ampliﬁcation of cDNA ends),and full-length PnGC-1 cDNA ampliﬁcation.PnGC-1fragment production by PCR.Total RNA was isolated from green cotyledons of P.nil seedlings and then the mRNA was puriﬁed using an Oligotex mRNA Mini Kit (Qiagen).Theﬁrst-strand cDNA for RT-PCR was synthe-sized with RevertAid M-MuLV RT(Fermentas)following the manufacturer’s instructions.Degenerate oligonucleo-tide primers50-TGG GA(C/T)TGC GG(C/T)CT(C/T) GCT-30and50-ACA TAT(C/T)AC(A/G)AC ATA(A/ G)TG(A/C/T)CC-30,corresponding to the conservedamino acid sequences WDCGLA and GHYVVIC,respec-tively,were designed for two conserved regions located in the regulatory domain of previously cloned putative plant GCs.The database accession numbers are as follows: Arabidopsis thaliana(AY118140,AAM51559),Hordeum vulgare(ABD18447),Zea mays(DQ372067),and Triticum aestivum(DQ372070).PCR was performed with50ng of cDNA and100ng of degenerate primers.PCR parameters consisted of50s at95°C for denaturing,50s at53°C for annealing,and50s at74°C for extension for35cycles,and aﬁnal extension step of7min at74°C.PCR products were separated on a 1.3%(w/v)agarose gel,eluted,and sequenced.30RACE of PnGC-1.30-RACE-ready cDNA synthesis was performed with the BD SMART RACE cDNA Ampliﬁcation Kit(BD Biosciences Clontech).The mRNA from total green cotyledons’RNA was puriﬁed and then reverse-transcribed with the30-RACE CDS Primer A [50-AAG CAG TGG TAT CAA CGC AGA GTA C(T)30VN-30,N=A,C,G,or T and V=A,G,or C].30 RACE was performed with the Advantage2Polymerase Mix(Clontech).The PCR reaction was performed by using a GSP(gene-speciﬁc primer)1(50-TTG AGG AAT TGG CAC AGT CCT GCT CT-30)and Universal Primer A Mix (UPM;Long:50-CTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA TCA ACG CAG AGT-30;Short: 50-CTA ATA CGA CTC ACT ATA GGG C-30)under the following conditions:94°C for30s,68°C for30s,and72°C for2min for35cycles.The PCR product was puriﬁed and cloned into a pTZ57R vector(Fermentas)for sequencing.Full-length PnGC-1cDNA ampliﬁcation.An alignment of sequences obtained from degenerate GSP1primers and a search of the NCBI EST database was used to predict a full-length PnGC-1cDNA.The sequence of50cDNA end was found in the dbEST of Ipomoea nil(synonym Phar-bitis nil)8-day-old seedling shoots(gi74391850).Thus, two PCR primers PnGC-1(50-GGT CCC AGT TTT GCA ACT TT-30)and PnGC-2(50-CAA AAT CAG TCA ACC CAG CA-30)were designed for the ampliﬁcation of full-length cDNA.The mRNA from green cotyledons was puriﬁed and reverse transcribed with the SuperScript III Reverse Transcriptase(Invitrogen)to obtain a PCR tem-plate.PCR was performed in a total volume of50l l reaction solution containing5l l109buffer A(minus Mg2?),2.5l l50mM MgCl2,1l l5mmol/l l each of dNTPs,1l l10l mol/l l PnGC-1,1l l10l mol/l l PnGC-2, 1%(v/v)DMSO,2l l cDNA,and1unit Yellow PfuPlus DNA polymerase(Eurx)using the following protocol: 95°C for5min followed by30cycles of95°C for30s, 61°C for30s,and72°C for1min,with aﬁnal extension of 7min at72°C.The nucleotide sequence of PnGC-1 reported here is available in GenBank under the accession number DQ672602.RNA Extraction and Reverse TranscriptionSamples were quick-frozen in liquid nitrogen and ground to powder by mortar and pestle.Total RNA was isolated from various organs(cotyledons,roots,hypocotyls)with the GeneMATRIX Universal RNA Puriﬁcation Kit(Eurx) according to the manufacturer’s instructions.Prior to reverse transcription,RNA samples were treated with RNase-free DNaseI(Fermentas)and then reverse tran-scribed with the MMLV RT enzyme(Epicentre)in20l l at 37°C for1h.Semiquantitative PCRRT-PCR analysis was performed to analyze the expression of the PnGC-1gene.PCR was conducted at the linearity phase of the exponential reaction for each gene.The gene-speciﬁc primer pairs were as follows:for PnGC-1,forward primer:50-TGA ACG TTC GTC TCA ACT GC-30and reverse primer:50-ACC GAC CAA AGC AAA CTC A-30; and for actin4gene(ACT),forward primer:50-GAA TTC GAT ATC CGA AAA GAC TTG TAT GG-30and reverse primer:50-GAA TTC CAT ACT CTG CCT TGG CAA TC-30.Ampliﬁcations were performed in a thermocycler programmed for30cycles of30s at94°C,45s at60°C, and45s at72°C.The actin4expression level was used as a quantitative control.Expression and Puriﬁcation of GST-PnGC-1Fusion ProteinThe full-length PnGC-1cDNA was ampliﬁed by PCR.The PCR product was veriﬁed by DNA sequencing and intro-duced into the plasmid pTZ57R/T(Fermentas).For expression of the GST-PnGC-1in bacteria,the PnGC-1ORF (open reading frame)was cut from pTZ57R/T and inserted into the pGEX-6P2(GE Healthcare)expression vector at Not1and Sal1restriction sites.The E.coli BL21strain, transformed with the resulting plasmid,was used to produce the GST-tagged protein.The expression of fusion protein was induced by the addition of isopropyl b-D-thiogalacto-side(IPTG)to aﬁnal concentration of1mM and incubation at24°C for3.5h.The bacteria cells were collected by centrifugation,suspended in lysis buffer[50mM Tris–HCl (pH8.0),150mM NaCl,5mM EDTA,0.5%(v/v)NP-40, 1mM PMSF,and0.2mg ml-1lysozyme],and disrupted by sonication.The soluble fraction was separated by centrifu-gation at12,000g for10min at4°C and the GST-tagged proteins were adsorbed onto glutathione–Sepharose4B beads(GE Healthcare).After washing the column with buffer containing50mM Tris–HCl(pH8.0)and150mM NaCl,the GST-PnGC-1complex was either eluted with 10mM glutathione in50mM Tris–HCl(pH8.0)or PnGC-1was released from the fusion protein by proteolytic cleavage of the protein with PreScission protease(GE Healthcare) following the manufacturer’s instructions.For a control expression,the pGEX-6P2vector was used or GST alone was puriﬁed as described above.Electrophoresis and Western Blotting AnalysisThe homogeneity and purity of eluted protein fractions were analyzed by12%(v/v)SDS–PAGE and gels were stained with Coomassie Blue.For Western blotting analysis,pro-teins resolved by SDS–PAGE were transferred onto poly-vinylideneﬂuoride(PVDF)membranes(Hybond-P,GE Healthcare)by the semidry system(BioRad)in25mM Tris,192mM glycine buffer(pH8.3)with20%(v/v) methanol.After blocking in TBS buffer(20mM Tris, 100mM NaCl)containing3%(w/v)nonfat dry milk,the membrane was incubated with polyclonal anti-GST anti-bodies(1:10,000)(GE Healthcare).The Western blots were visualized with horseradish peroxidase-conjugated sec-ondary anti-goat IgG antibodies(1:30,000)(Sigma-Aldrich) and the blots were detected using a chemiluminescence kit (ECL plus)following the manufacturer’s instructions(GE Healthcare).Determination of cGMP and cAMP ConcentrationThe concentration of cGMP or cAMP was measured with a speciﬁc[3H]-radioimmunoassay kit(Amersham Pharmacia Biochem).Analyzed samples were added to scintillation vials containing scintillation cocktail and counted in a liquid scintillation counter(Wallac1407).Data are expressed as pmol cGMP g-1fresh tissue or pmol cyclic nucleotide(cGMP or cAMP)min-1mg-1protein.All measurements were performed in duplicates and repeated three times.Guanylyl Cyclase ActivityGuanylyl cyclase activity was determined as described previously by Kamisaki and others(1986)and Szmidt-Jaworska and others(2008b)by estimating the rate of cGMP formation.The analyzed fractions(containing PnGC-1)were assayed for guanylate cyclase activity in a ﬁnal volume of100l l at30°C for10min with gentle shaking.The incubation buffer contained50mM Tris–HCl (pH7.5),4mM MnCl2,1mM GTP,1mM DTT,and 0.5mM isobutylmethylxanthine.The reaction was termi-nated by the addition of96%(v/v)ethanol.The samples were shaken for5min,incubated on ice for10min,and then centrifuged at13,000g for10min.Supernatants were collected and lyophilized.The concentration of the formed cyclic GMP was determined by radioimmunoassay as described above.The enzyme activity was deﬁned as the amount of cGMP produced by1mg of protein per minute.The effect of temperature on the activity was evaluated with the standard activity assay at pH7.5and different temperatures in the20–37°C range.Kinetic parameters(K m and V max)were determined from rate measurements(1–20min)using GTP concentrations from0.1to3.0mM.The data were processed following the classical Lineweaver-Burk-type plots.All the measure-ments were performed in three replicates.Moreover,ATP (0.1–3mM)was used to analyze the speciﬁcity of the identiﬁed enzyme.The inhibitor’s and activators’effects were determined by comparing the enzymatic reaction rates obtained when 1mM SNP and1mM NS2028were added in the presence or absence of divalent cations(Mn2?or Mg2?).ResultsMolecular Cloning of PnGC-1The PnGC-1gene was isolated using degenerated PCR primers that were derived from conserved sequences of previously cloned GCs of other plant species.RT-PCR ampliﬁcation was performed on mRNA from cotyledons.A DNA fragment of the expected size was recovered from the agarose gel,TA-cloned into the plasmid vector,and sequenced.The complete coding sequence of the GC gene, designated as PnGC-1,was obtained using RACE-PCR. The full-length PnGC-1cDNA contains start and stop codons and consists of a861-bp ORF which encodes a286-amino-acid peptide(Fig.1)with a theoretical molecular mass of32.3kDa and a pI of5.7.The full-length sequence of PnGC-1has been placed into GenBank under accession number DQ672602.A search of the protein database using BLASTP revealed that the deduced amino acid sequence of PnGC-1 exhibited homology to other GCs,including S.lycopersi-cum(61%identity),A.thaliana AtGC-1(56%identity),T. aestivum(52%identity),and O.sativa(57%identity) (Fig.2).PnGC-1contains all typical features of other plant GC(Fig.3).It contains a variable catalytic motif in its N-terminal region and highly conserved C-terminal region.The catalytic domain exhibited high conservation of glycines(G,positions32and39)and arginine(R,position 21),all responsible for interaction with the phosphate group in GTP via a water molecule.Moreover,it possesses cysteine(C,position13)responsible for recognition of the guanine.The glutamic acid and/or aspartic acids(E and D, position25and/or51)are believed to bind essential metal ions(Mn2?or Mg2?).The asparagine(N,position33)and lysine(K,position18)are likely responsible for transition-state stabilization.It is possible to ﬁnd putative myris-toylation sites in both N and C terminals of the predicted GC protein.To assess the properties of PnGC-1,the full-length gene was expressed in E.coli BL21as a glutathione S-trans-ferase (GST)-fusion protein.The PnGC-1was cloned into pGEX-6P2and the recombinant PnGC-1was expressed in E.coli using the GST-fusion system.When the protein expression was induced by addition of IPTG,GST-PnGC-1emerged as a clear main band with a molecular mass of 56kDa,which was not observed in control fractions (empty pGEX vector or pGEX-PnGC-1without IPTG application)(Fig.4a).When a sample of this puriﬁed protein was digested with PreScission protease,one main 32-kDa band appeared,corresponding to PnGC-1(Fig.4a).Western blot analysis showed that an anti-GST antibody reacted with the 56-kDa peptide as well as with GST(26kDa)(Fig.4b),demonstrating that the induced protein was GST-PnGC-1.The possibility that the expression of pGEX-PnGC-1forms an active enzyme was analyzed by in vivo and in vitro analyses of enzyme activity.When an equal amount of cells was extracted and assayed for cGMP,a 2.5-fold increase was observed in extracts from transformed,induced bacteria compared with extracts from a nonin-duced cell and the control (empty pGEX vector)(Fig.5).Enzymatic analysis performed with the supernatant also revealed a positive reaction.The supernatant after IPTG treatment had a GC activity of about 0.18pmol min -1mg -1protein.Cyclic GMP,as a product of PnGC-1activity,was undetectable when IPTG was not applied (Fig.6a).To analyze the kinetic properties of puriﬁed PnGC-1,cGMP formation was determined in the presence of increasing GTP concentration (Fig.6b).A Lineweaver-Burk plot of the data revealed a K m value of 0.87mM GTP and V max of 78.1pmol min -1mg -1protein,suggesting proper folding of the catalytic domain.Moreover,the possibility that a recombinant protein might act as both an adenylyl and guanylyl cyclase was examined directly.However,in the case of PnGC-1,no associated adenylyl cyclase activity toward ATP was detected (Fig.6b).The effect of temperature on PnGC-1activity was determined within a range of 20–37°C,and the optimum temperature was found to be approximately 30°C (Fig.6c).As was shown in Fig.6d,PnGC-1essentially requires divalent metal ions for its enzymatic activity and Mn 2?isFig.1Nucleotide and deduced amino acid sequences of guanylyl cyclase from Pharbitis nil .Numbering starts at the putative transla-tion initiation codon for nucleotide (left margin).Stop codon is marked by anasteriskFig.2A rooted phylogenetic tree based on the nucleotide sequences illustrates the relationship between the cloned plant GC.The percentage of identical (%Id)and similar (%Sim)residues is presented.The phylogenetic tree was constructed using ClustalW in GenBank.The EMBL database accession numbers are as follows:P.nil (ABG67691),A.thaliana (AAM51559),Z.mays (DQ372067),S.lycopersicum (ABK15531),G.max (ABK15530),H.vulgare (ABD18447),T.aestivum (ABD18449),and O.sativa (ABD18448)the most favorable.Apart from metal ions,the inﬂuence of SNP (as a NO donor and animal sGC stimulator)and NS 2028(as a GC inhibitor)on the activity of recombinant PnGC-1was tested.Application of SNP did not alter the enzyme activity,whereas NS 2028caused a signiﬁcant (4.5-fold)reduction in the production of cGMP.Expression Analysis in Vegetative OrgansTo investigate changes in the PnGC-1expression level,plant organs’speciﬁcity was analyzed ﬁrst.A semiquanti-tative RT-PCR technique was used to monitor the tissue-speciﬁc transcript levels of PnGC-1.Total RNAwasFig.3A comparison of the amino acid sequences ofputative guanylyl cyclases from various plants.Black and shaded backgrounds indicate regions of identity and consensus,respectively,between eight GC from various plants.The alignment was generated using the VECTOR program.Dashes represent sequence gaps to allow for maximum alignment.In the catalytic domains the glycine-rich motifs are indicated as r ;the PP i -binding residue is indicated as ;;the guanine-binding residue is indicated as r ;the metal ion-bindingresidues are indicated as d ;the amino acids responsible for transition state stabilization are indicated as H .The arrows indicate the amino acid residues corresponding to the degenerate oligonucleotides.The accession numbers of the GC sequences are indicated in Fig.2isolated from cotyledons,stems,and roots of P.nil seed-lings grown either in continuous white light or treated with darkness.As shown in Fig.7,PnGC-1mRNA wasdetected in all organs,but the highest expression pattern was observed when plants were grown in darkness.In these plants the expression level was higher in hypocotyls and roots than in cotyledons.The transcript levels of PnGC-1were found to be downregulated by light.The most pro-nounced decrease (2-and 2.5-fold)was found in cotyle-dons and hypocotyls,respectively.The ampliﬁcation of the actin4gene in the corresponding samples was used to determine the availability of relatively intact RNA samples and to normalize the quantiﬁcation of the target (PnGC-1)transcript.The level of actin4mRNA did not show any statistically signiﬁcant variation,thereby validating the results obtained.Expression Analysis Under Photoperiodic Conditions To determine the relationship between PnGC-1expression and the light/dark treatment,we followed changes in PnGC-1expression during two different light/dark regimes.To analyze the expression pattern of PnGC-1during short-day conditions (8/16h photoperiod),we compared mRNA populations extracted from cotyledons that either had or had not been exposed to a 16-h-long night.Total RNA was isolated from cotyledons at 1-h intervals,and the levels of mRNA were determined by RT-PCR analysis (Fig.8a).The normalized PnGC-1to actin4transcript ratio changed during the long night.The transcript level reached three peaks at the 3rd,7,and 11th hour after moving plants to the darkness and then fell during the subsequent dark period.In contrast,in the seedlings that were kept in light,the level of GC mRNA remained at a basallevel.Fig.4Puriﬁcation and characterization of recombinant PnGC-1.The full-length PnGC-1reading frame was expressed in E.coli as a GST-fusion protein.Puriﬁcation was monitored by SDS-PAGE followed by Coomassie Brillant Blue staining (a )or Western blot analysis with anti-GST antibody (b ).a ne 1is protein molecular mass marker (kDa).Lane 2is the supernatant from noninduced E.coli ne 3is the supernatant from IPTG-induced E.coli ne 4is the recombinant GST-PnGC-1puriﬁed using a glutathione afﬁnity ne 5is a PnGC-1after digestion with PreScission ne 6is GST protein.b Western nes 2and 3are supernatants from noninduced (-)and IPTG-induced (?)culture of E.coli transformed with ne 4is a puriﬁed ne 5is a recombinant ne 6is a GSTproteinFig.5Content of endogenous cGMP in E.coli cells determined with [3H]cGMP radioimmunoassay system.The control (C)is the concentration of cGMP in the whole-cell E.coli extract containing empty pGEX 6P2after IPTG addition.?and -show the value of cGMP in IPTG-induced or noninduced extracts of E.coli containing the construct (pGEX-PnGC-1).All assays were performed in duplicate and each experiment was repeated three timesIn addition,an examination was undertaken to discover whether light also affects the mRNA level in cotyledons already exposed to the darkness.Seedlings of P.nil exposed to a 16-h-long night were treated with R or FR in the middle of the night.The interruption of the darkness by R reduced the level of PnGC-1mRNA rapidly,and then a renewed increase was observed (Fig.8b).FR light alone did not reduce the PnGC-1mRNA level (data not shown).To determine whether FR could reverse the effect of R,the same set of experiments were performed and R and FR were added in the middle of the 16-h-long night,respec-tively.The effectiveness of R followed by FR was still very high (data not shown);therefore,FR was not able to reverse the effect caused by R.Experiments conducted in traditional conditions for cultivation of P.nil seedlings did not distinguish between the phytochrome types involved.This was the reason why special photoperiodic conditions for cultivation of seed-lings were used (72-h-long darkness,24-h-long white light of low intensity,and 24-h-long night with or without FR before the night).In such conditions,moving plants to the darkness changed the amount of the transcript,reaching a maximal level at 3,7,and 12h of the night and decreasing during the subsequent dark period (Fig.9).When FR treatment preceded the night,the oscillations did not occur.However,when FR and subsequent R were given prior to the 24-h-long night,the PnGC-1expression pattern was restored (data notshown).Fig.6Biochemical properties of recombinant PnGC-1.Values are the means of threereplicates.Bars represent SE.a Guanylyl cyclase activity toward Mn-GTP.C is the enzyme activity in thesupernatant from noninduced and IPTG-induced E.coli cells containing empty pGEX 6P2.S is the enzyme activity in the supernatant from noninduced and IPTG-induced E.coli cells transformed with pGEX-PnGC-1.b Determination ofrecombinant PnGC-1cyclase activity in response toincreasing concentration of substrate (GTP or ATP,in the range of 0.1-3mM).c Effect of temperature on PnGC-1activity.d Comparison of enzyme activity after application of various substances (divalent cations:Mn 2?and Mg 2?;SNP as a NO donor and potentsoluble GC stimulator;NS2028as a potent soluble GC inhibitor)。

how to read Literature

看文献的学问结合看到的一些真知灼见，加上我这次历时4周的prelim（preliminary，准备；预赛；初步措施）考试(精读+粗读，怎么也得两三百篇吧)，我也来也谈谈看文献。

其实看文献也是一种学问，因为掌握多少知识并不重要，至少不是最关键的，知道怎样去掌握知识才是最关键的。

千万不要小看这个，这里面可大有文章呢。

看文献最关键的在于要自己明白这篇文献到底说了什么，bottom line是什么，到底哪一点是有新意的(才开始的时候不容易做到)。

看文献的大忌就是读的时候似乎都明白，读完后就不知所云。

在我看来，以下几点：1. 不管是粗读还是精读，读完后，都要自己做个brief的总结，take home message(重点总结)是什么，文章着重是解决什么问题，有什么巧妙的地方。

什么，不知道？打回去重读，看abstract、disscusion、conclusion，一般这种信息不会很多的，也不是要死记硬背的那种，如果你发现你很费劲才能记住，那说明你没抓住重点。

我的经验是：如果看几遍都毫无进展，那说明要么是你的阅读太慢了(-->至少浏览每段的首句，强迫自己集中精力读快，你会发现经过一段时间你是能够做到的)，要么是你的背景知识不够(-->追踪参考文献，我粗略估计了一下，一般在2～3篇文章的references list（reference，参考；参照；涉及，提及；参考书目；引用）里面就会出现一篇很好的review（review，回顾；复习；评论呢；检讨；检阅），接下来请看suggestion 2)。

2. 如果是新手才入门的，或是想了解新的领域，最好的办法就是先看大牛们的reviews(如果你不知道谁是大牛，那就在pubmed里面搜这个领域的关键词+review type，或是在同等类型文章后面的list比对，出现频率较高的)，看review 的诀窍是少而精。

不求一遍就看懂(如果那样你都可以写review 了)，仔细翻查references，如果你能潜心钻研其中的2/3，那么恭喜你，你已经入门了；当然如果有认识的朋友在相关领域的，不妨交流交流，可以省去你走很多弯路的。

（完整版）名词解释分子生物学

（完整版）名词解释分子生物学分子生物学名词解释基因组，Genome，一般的定义是单倍体细胞中的全套染色体为一个基因组，或是单倍体细胞中的全部基因为一个基因组半保留复制（semiconservative replication）：一种双链脱氧核糖核酸（DNA）的复制模型，其中亲代双链分离后，每条单链均作为新链合成的模板。

因此，复制完成时将有两个子代DNA分子，每个分子的核苷酸序列均与亲代分子相同，半不连续复制(Semi-ondisctinuousreplication)。

是指DNA复制时，前导链上DNA的合成是连续的，后随链上是不连续的，故称为半不连续复制。

dNTP，deoxy-ribonucleoside triphosphate（脱氧核糖核苷三磷酸）的缩写。

是包括dATP, dGTP, dTTP, dCTP,dUTP等在内的统称，N是指含氮碱基，代表变量指代A、T、G、C、U等中的一种。

在生物DNA、RNA合成中，以及各种PCR（RT-PCR（reverse transcription PCR）、Real-time PCR）中起原料作用。

转座子是一类在细菌的染色体，质粒或噬菌体之间自行移动的遗传成分，是基因组中一段特异的具有转位特性的独立的DNA序列.多顺反子（polycistronicmRNA)在原核细胞中，通常是几种不同的mRNA连在一起，相互之间由一段短的不编码蛋白质的间隔序列所隔开，这种mRNA叫做多顺反子mRNA。

这样的一条mRNA链含有指导合成几种蛋白质的信息。

基因表达：(gene expression)是指生物基因组中结构基因所携带的遗传信息经过转录、翻译等一系列过程，合成特定的蛋白质，进而发挥其特定的生物学功能和生物学效应的全过程外显子(expressed region)是真核生物基因的一部分，它在剪接(Splicing)后仍会被保存下来，并可在蛋白质生物合成过程中被表达为蛋白质。

Molecular cloning, characterization, and immunolocalization of two lactate dehydrogenase homologous

ORIGINAL PAPERMolecular cloning,characterization,and immunolocalization of two lactate dehydrogenase homologous genesfrom Taenia soliumWuying Du&Fengyu Hu&Yabo Yang&Dong Hu&Xuchu Hu&Xinbing Yu&Jin Xu&Jialin Dai&Xinjiang Liao&Jiang HuangReceived:1November2010/Accepted:2February2011#Springer-Verlag2011Abstract Two novel genes encoding lactate dehydrogenase A(LDHA)and B(LDHB)homologues,respectively,were identified from the cDNA libraries of adult Taenia solium (T.solium).The two deduced amino acid sequences both show more than50%identity to the homologues for Danio rerio,Xenopus laevis,Schistosoma japonicum,Sus scrofa, Homo sapiens,et al.The identity of the amino acid sequence between Ts LDHA and Ts LDHB is57.4%,and that of the nucleotide sequence is61.5%.Recombinant Ts LDHA homologue(r Ts LDHA)and Ts LDHB homologue (r Ts LDHB)were expressed in Escherichia coli BL21/DE3 and purified.Though there were some differences in the sequence,the two LDH isozyme homologues show similarity in the conserved LDH domain,topological structure,primary immunological traits,localization on the tegument of T.solium adult,and partial physicochemical properties.The linear B-cell epitope analysis of Ts LDHA and Ts LDHB discovered a Ts LDHA specific epitope.The purified r Ts LDHA and r Ts LDHB could be recognized by rat immuno-sera,serum from swine,or a patient infected with T. solium,respectively,but Western blot analysis showed cross-reactions,not only between these two LDH members but also with other common human tapeworms or helminths. The results suggested that the two LDH homologues are similar in the characteristics of LDH family,and they are not specific antigens for immunodiagnosis.IntroductionTaenia solium is prevalent in most developing countries and has been spreading to developed countries as well by massive human migration.Approximately50million people worldwide were infected with T.solium(Sciutto et al.2000;Aguilar-Diaz et al.2006),20million people were infected with T.solium cysticerci(Bern et al.1999), and50,000deaths occur from this parasite every year (Mafojane et al.2003).Taeniasis and cysticercosis remain the worldwide important public health problems which need to be solved.It is known that lactate dehydrogenase(LDH)is a terminal glycolytic enzyme,which catalyzes the inter-conversion of pyruvate and lactate in the presence of the nicotinamide adenine dinucleotide coenzyme.The ex-pression of LDH isozymes composed of lactate dehydro-genase A(LDHA)and lactate dehydrogenase B(LDHB) subunits have obvious species and tissue specificities, which adapt to their biochemical properties and physio-logical function(Rheede et al.2003;Alcazar et al.2000; Imagawa et al.2006;Laughton et al.2007).The enzymatic activity of the LDH of Babesia bovis(Borket W.Du:J.Dai:X.Liao:J.Huang(*)Department of Parasitology,Guiyang Medical College,Guiyang,Chinae-mail:Huang31222@F.Hu:D.HuInstitute of Infectious Diseases,Guangzhou No.8People’s Hospital,Guangzhou,ChinaY.YangDepartment of Dermatology,Sun Yat-sen Memorial Hospital,Sun Yat-sen University,Guangzhou,ChinaX.Hu:X.Yu:J.XuDepartment of Parasitology,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou,ChinaParasitol ResDOI10.1007/s00436-011-2285-8al.2004),Toxoplasma gondii(Dando et al.2001), Spirometra mansoni(Kwak et al.1996),Clonorchis sinensis(Yang et al.2006),Schistosoma japonicum(Lu et al.2007a,b),and Taenia asiatica(Huang et al.2008)is inhibited by gossypol or anti-parasite drugs such as praziquantel and artemether which suggested that LDH is a drug target of anti-helminth drugs.The immunohistochemistry demonstrated that the LDH of C.sinensis,S.japonicum,and tica is a membrane-associated protein,located at the surface of the digestive tract and tegument in the C.sinensis adult or the tegument of tica adult and the embryonic mem-brane of the oncosphere(Huang et al.2008;Lu et al. 2006;Huang et al.2009),respectively.LDH is one of the antigens in S.japonicum(Lu et al.2007a,b)or tica (Huang et al.2009);the LDH of the malarial parasite released into the blood is a diagnostic antigen which had been applied in a diagnostic kit(Garcia et al.2003).All the works on LDH of parasites mentioned above strongly suggested that membrane-associated LDH is an important target for new anti-parasite drugs and a candidate molecule for vaccine development or diagnosis.The LDHA from tica has been characterized,but the homologue in another important Taenia,T.solium,has not been described.In the present study,we identified two expressed sequence tags(ESTs)(clones Ts0_001458and Ts0_001711)encoding LDHA and LDHB homologues by online BLASTx analysis from the complementary DNA(cDNA)library of T.solium adult we constructed and,for the first time,comparatively described their sequence,expression,and immunolocaliza-tion.This study might facilitate the understanding of the property of energy metabolism of T.solium and its potential application in diagnosis,drug design,and vaccine develop-ment for T.solium.Materials and methodsIdentification and bioinformatics analysisThe cDNA library of adult T.solium was constructed by the method of switching mechanism at5′end of the RNA transcript.From the1,981ESTs sequenced,we have obtained1,070unigenes.All of these cDNA sequences and their deduced protein sequences were analyzed using the basic local alignment search tool(BLAST)at the National Center for Biotechnology Information(NCBI) website().From the library,two ESTs(clones Ts0_001458and Ts0_001711)were identified as complete homologues of LDHA and LDHB by BLASTx.The two sequences were submitted to GenBank(access numbers GU571142,GU571143).The complete encoding sequences(CDS) of T.solium LDHA and LDHB(Ts LDHA and Ts LDHB) were determined by the Open Reading Frame(ORF) Finder program at the NCBI web server(http://www.ncbi. /projects/gorf/).The cDNA and deduced amino acid sequences of Ts LDHA and Ts LDHB were analyzed by Vector NTI software package.Protein analysis was done with Proteomics and sequence analysis tools(/tools),and linear B-cell epitopes were analyzed by Bepipred Linear Epitope Predic-tion(/tools/bcell/)of the IEDB Analysis Resource.Cloning,expression,and purificationThe complete coding sequences of Ts LDHA and Ts LDHB were amplified by polymerase chain reaction(PCR)using the plasmid of Ts0_001458and Ts0_001711clones as templates,respectively.The forward primer of the Ts LDHA gene was5′-TAT CCA TGG AAA TGC CTG GTG GGG GTT TG-3′,and its reverse primer was5′-GCG CTC GAG CCA CTT GAT GTC TGC GAT AAT-3′with Nco I and Xho I as restriction enzyme sites(shown as the underlined parts),respectively.And the forward primer of the Ts LDHB homologue was5′-GGC CAT ATG ATG GCT GAA CAT TCT ATC C-3′,and the reverse primer was5′-AGA CTC GAG CCA TTT GAT ACC AGA AGT AG-3′with Ned I and Xho I sites to the5′ends,respectively.The amplifica-tion condition was94°C for1min,61°C(Ts LDHA)or 56°C(Ts LDHB)for1min,and72°C for1.5min,for35 cycles using ExTaq polymerase(Takara Shuzo Co.,Kyoto, Japan).After digestion with corresponding endonucleases and purification,the products of PCR were recombined into the pET-28a(+)expression vector(Novagen)with the coding region in frame with the6×His tag.The correct recombinant plasmids(identified by DNA sequencing) were expressed in E.coli BL-21strain(DE3)in Luria–Bertani medium containing50μg/ml kanamycin.The expression of the recombinant fusion proteins was induced by IPTG at a final concentration of0.6mM for5h at28°C. The bacterial cells were collected by centrifugation at4°C, and the recombinant fusion proteins were purified with His Bind Purification kit(Novagen)according to the manufac-turer's instructions.The imidazole in the elution buffer was removed by dialysis using phosphate-buffered saline (PBS,pH7.4).The expression and purity of recombined Ts LDHA(r Ts LDHA)and Ts LDHB(r Ts LDHB)were identified by12%sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS–PAGE)and visualized by Coomassie brilliant blue G-250.The final concentrations of the two purified recombinant proteins were determined by the method of Bradford on nucleic acid/protein analyzer(Beckman,USA).Parasitol ResThe expressions of the two recombinant proteins were also identified by Western blot with mouse anti-his tag monoclonal antibody as the probe.The lysate of E.coli with pET28a(+)after induction,lysate of E.coli with pET28a(+)-Ts LDHA and pET28a(+)-Ts LDHB before and after induction,supernatant and precipitant of the lysate of E.coli with pET28a(+)-Ts LDHA and pET28a(+)-Ts LDHB after induction were subjected to SDS–PAGE(12%gel) and electrotransferred to polyvinylidene fluoride(PVDF) membrane(Qbiogene).Then,the membrane was blocked with5%(w/v)skim milk powder in PBS(pH7.4)at4°C overnight and incubated with the mouse anti-his tag monoclonal antibody(1:3,000dilution;Invitrogen)at 37°C for2h.After being washed,the membrane was incubated with goat anti-mouse immunoglobulin G(IgG)-conjugated HRP(1:5,000dilution;Boster Co.)at37°C for 1h,and the HRP was visualized by diaminobenzidine (DAB)(Boster Co.)substrate solution.Preparation of anti-serum and collection of infected serumThe r Ts LDHA and r Ts LDHB were emulsified with Freund's adjuvant(complete adjuvant for the first injection,incom-plete adjuvant for the booster)and injected subcutaneously to Sprague–Dawley rats three times.Each animal was given 200μg of the recombinant protein at the first injection and 100μg of protein in two booster injections at2-week intervals.Two weeks after the last injection,sera were collected as the anti-serum against r Ts LDHA and r Ts LDHB.The titers of anti-r Ts LDHA and anti-r Ts LDHA immune sera were both above1:409,600,determined by enzyme-linked immunosorbent assay.At Yajiang County in Sichuang Province,an epidemic area of T.solium,the adults of T.solium were obtained from patients discharging gravid proglottids with oral administration of arecaline-magnesium sulfate agent.The adults were morphologically identified as T.solium by experienced specialists.Sera from the patients were isolated and stored at−80°C.Live eggs collected from a gravid segment were used to infect pigs.After6months, the infected pigs were slaughtered to isolate serum and cysticerci from the liver.The sera from patients infected with Taenia saginata and tica were provided by the Department of Parasitology in Guiyang Medical College,and the sera from patients infected with C.sinensis and S.japonicum were provided by the laboratory of the Department of Parasitology in Zhongshan Medical College,Sun Yat-sen University.Western blot analysisThe r Ts LDHA and r Ts LDHB(0.5–0.7μg per lane)were subjected to SDS–PAGE(12%gel)and electrotransferred to PVDF membrane(Qbiogene).Then,the membrane was blocked with5%(w/v)skim milk powder in PBS(pH7.4) at4°C overnight,incubated with the primary antibody at 37°C for2h;the rat anti-r Ts LDHA and anti-r Ts LDHB serum(1:1,000dilution),serum from swine infected with T. solium(1:200dilution),and serum from patients infected with T.solium,T.saginata,tica,C.sinensis,and S. japonicum(1:200dilution)were employed as primary antibodies,respectively.Pre-immune rat serum and normal serum from swine and humans were used as the corresponding control.After washing,the membrane was incubated accordingly with goat anti-rat IgG-conjugated HRP(1:2,000dilution;Boster Co.),peroxidase-labeled goat anti-swine IgG(γ)(1:500dilution;Bethyl,USA), and goat anti-human IgG-conjugated HRP(1:2,000dilution; Boster Co.)at37°C for1h,and the HRP was visualized by DAB(Boster Co.)substrate solution. Immunolocalization in T.solium adultSeveral segments of T.solium adult worms fixed with 10%neutral formalin were embedded with paraffin and sliced in4-μm thickness.After deparaffinization,freshly prepared1%sodium borohydryde in1%disodium hydrogen phosphate monohydrate was applied to the sections for10min to quench the autofluorescence. Sections were then blocked with normal goat serum blocking solution(Boster Co.)at4°C overnight.The rat anti-serum against r Ts LDHA and r Ts LDHB was employed as the primary antibody,respectively,and pre-immune rat serum was used as negative control;the sections were incubated in the primary antibody for2h at37°C.After washing,the sections were incubated in Alexa-Fluor555 dye-conjugated goat anti-rat IgG(H+L)(Molecular Probes,Invitrogen)(1:400dilution)for1h at37°C. Washed again,the sections were imaged by fluorescence microscope(Olympus IX61)and a computer.ResultsBioinformatics analysis of Ts LDHA and Ts LDHBThe EST clones Ts0_001458and Ts0_001711with com-plete encoding sequence of Ts LDHA and Ts LDHB were identified from an adult T.solium cDNA library through BLASTx.The two deduced amino acid sequences had more than50%identity to LDHA and LDHB,respectively,with other species,such as Danio rerio,Xenopus laevis,S. japonicum,Sus scrofa,Homo sapiens.The degree of amino acid sequence identity between Ts LDHA and Ts LDHB is 57.4%,and that of nucleotide sequence is61.5%by Vector NTI suite8.Both sequences contain a complete ORFParasitol Rescomprised of 993base pairs,encoding a putative protein of 331amino acids.The molecular weight (MW)of Ts LDHA was predicted to be 35.46kDa,the theoretical isoelectric point (pI)was 7.09,and the corresponding parameters of Ts LDHB were 35.58kDa and 7.21.The estimated half-lives of Ts LDHA and Ts LDHB were both more than 10h in E.coli ;the two isozymes possess similar secondary structures composed mainly of α-helix and random coil.No N-terminal signal peptide was found in either Ts LDHA or Ts LDHB.The sequence alignments of the two EST cDNA showed that the conserved LDH domains both located at aa190-aa195(EGHGDS)in Ts LDHA or Ts LDHB predicted by Motifscan and InterProscan and the histidine residue (H)located at aa193is one of the LDH active sites.Predict-Protein predicted there are three transmembrane regions in both Ts LDHA (T1,aa29–aa39;T2,aa129–aa144;T3,aa250–aa 259)and Ts LDHB (T1,aa27–aa47;T2,aa128–aa145;T3,aa247–aa 264).The characteristic of these transmembrane regions is α-helix.Though three transmem-brane regions of Ts LDHA are all shorter than the counterparts in Ts LDHB,their positions are very similar to that of Ts LDHB,and most amino acids in each corresponding transmembrane region are the same.By Bepipred Linear Epitope Prediction,there were five main linear B-cell epitopes predicted in Ts LDHA (aa13–aa20,aa80–aa87,aa190–aa199,aa215–aa228,aa307–aa314),which were named from E1to E5in order,and four (from E1to E4)in Ts LDHB (aa13–aa21,aa79–aa86,aa191–aa197,aa215–aa227).The positions of epitopes from E2to E4of Ts LDHA are similar to those of Ts LDHB,and partial amino acid residues in epitopes E2,E3,and E4are the same between two isozymes,especially the majority of amino acid residues of two E3epitopes containing the same LDH-conserved domain.But the epitopes E1and E5are verydifferent;there are no same amino acid residues in two E1epitopes,and in the region from aa307to aa314in Ts LDHB,corresponding to Ts LDHA E5,no epitope was predicted.The conserved LDH domains,transmembrane regions,and B-cell epitopes were demonstrated in Fig.1.Prokaryotic expression and purificationThe correctly constructed recombinant pET-28a(+)vector containing the Ts LDHA coding region was verified by sequencing.The r Ts LDHA and r Ts LDHB were expressed in E.coli BL21/DE3after induction by IPTG;their MWs were approximately 35.4and 36kDa (on polyacrylamide gel stained by 0.1%Coomassie brilliant blue R250),respectively,consistent with the prediction.The two sole proteins of r Ts LDHA (Fig.2a )and r Ts LDHB (Fig.2b )were obtained by affinity chromatography purification,and the expression level of recombinant proteins were 0.6mg (r Ts LDHA)(Fig.2a ,lane 7)and 0.8mg (r Ts LDHB)per milliliter culture (Fig.2b ;lane 7).Western blot with mouse anti-his tag monoclonal antibody as probe also indicated the successful expression and purification of r Ts LDHA and r Ts LDHB (Fig.3).Western blot analysisFigure 4shows the results of Western blot analysis.The purified r Ts LDHA and r Ts LDHB could be recognized by rat anti-r Ts LDHA and anti-r Ts LDHB immune sera,respec-tively (Fig.4a ;lanes 3,4),and both of the two purified recombinant proteins could be recognized obviously by swine serum (Fig.4a ;lanes 7,8)and patient serum (Fig.4a ;lanes 11,12)infected with T.solium in the Western blot analysis.Pre-immune rat serum and normal sera from swine and humans did not react with either r Ts LDHA (Fig.4a;Fig.1Alignment of thededuced amino acid sequence of Ts LDHA cDNA to that of Ts LDHB cDNA.The framed sequences from E1to E5were linear B-cell epitopes.The underlined sequences T1,T2,and T3represent threetransmembrane regions,and the conserved LDH domains are shown with italicized lettersParasitol Reslanes 1,5,9)or rTsLDHB (Fig.4a ;lanes 2,6,10).In addition,the Western blot analysis showed there were cross-reactions between r Ts LDHA and r Ts LDHB,and the two recombinant proteins showed obvious cross-reaction as with other helminths and did not react with the normal sera from healthy persons.Immunolocalization in T .solium adultUsing the rat anti-serum against the recombinant Ts LDHA (Fig.5a )and Ts LDHB protein (Fig.5c )as the primary antibody and fluorescence labeling goat anti-rat IgG as the second antibody,the reddish-orange fluorescence could be observed at the continuous tegument of adult T.solium as the arrows indicated,not in other internal tissues of T.solium .And the negative controls incubated with pre-immune rat serum did not show fluorescence (Fig.5b,d).DiscussionThe interesting property of parasite energy metabolism is anaerobic glycolysis;their energy production mainly depends on glycolysis.As a terminal enzyme of glycolysis,LDH is crucial for the survival of parasites,especially those inhabiting the host's intestine,and is highly expressed.This significant difference with its host has been considered as an important target of anti-parasite drugs.Actually,a series of investiga-tions have demonstrated that some antiparasitics such as artemether (Xiao et al.1999),praziquantel,levamisole,and benzimidazoles (V eerakumari and Munuswamyl 2000)act on the processes of glycogen metabolism including the reaction catalyzed by LDH,praziquantel,and gossypol that showed a high inhibition on the enzymatic activity of recombinant LDH from S.japonicum (Lu et al.2007a ,b );gossypol and its derivative have the function against malarial or bovine babesiosis with LDH as a potential chemotherapeutical target (Camemn et al.2004;Bork et al.2004;Conners et al.2005).As an important target of antiparasitics,characterization of structure and function is necessary to elucidate the pharma-cological mechanisms of anti-parasite drugs.As an important digestive duct helminth and harmful pathogen,T.solium and cysticercosis have always been the focus of human taeniasis studies.In our previous works,we characterized the LDHA of tica ,but the information about the LDH in T.solium is very few.It is known that LDH in mammals belongs to the isozymes family including six members composed of subunits A,B,and C encoded by three paralogous genes Ldh-A ,Ldh-B ,and Ldh-C ,respec-tively.In this study,we identified two paralogous genes of the LDH family from T.solium adult cDNA library.The deduced amino acid sequences show high identitytoFig.3Western blot analysis of the expression of r Ts LDHA and r Ts LDHB with mouse anti-his tag monoclonal antibody.Protein molecular weight markers (M );lysate of E.coli with pET28a(+)after induction (lane 1),lysate of E.coli with pET28a(+)-Ts LDHA (lane 2)or pET28a(+)-Ts LDHB before induction,and (lane 6)lysate of E.coli with pET28a(+)-TsLDHA (lane 3)or pET28a(+)-Ts LDHB (lane 7)after induction,supernatant (lanes 4,8)and precipitant (lanes 5,9)of lysate of E.coli with pET28a(+)-Ts LDHA or pET28a(+)-Ts LDHB afterinductionFig.2Analysis of expression and purification of r Ts LDHA and r Ts LDHB by 12%SDS –PAGE stained with Coomassie brilliant blue.a Shows the analysis of r Ts LDHA and b is the result of r Ts LDHB.Protein molecular weight markers (M ),lysate of E.coli with pET28a (+)before induction (lane 1)and after induction (lane 2),lysate of E.coli with pET28a(+)-Ts LDHA or pET28a(+)-Ts LDHB before induc-tion and (lane 3)after induction (lane 4),supernatant (lane 5),and precipitant (lane 6)of lysate of E.coli with pET28a(+)-TsLDHA or pET28a(+)-Ts LDHB after induction,lane 7,r Ts LDHA or r Ts LDHB after purificationParasitol ResLDHA or LDHB genes of previously reported homologues from other species.Bioinformatics analysis revealed that their structural characteristics are consistent with the properties of LDHA and LDHB and were theoretically identified as LDHA and LDHB genes of T.solium .Both deduced amino acid sequences have three transmem-brane regions,suggesting they are membrane proteins.Each transmembrane region is conserved in position and amino acid sequence in Ts LDHA and Ts LDHB.Immunolocalization of Ts LDHA and Ts LDHB demonstrated that both localize in the tegument of T.solium adult,which is similar with the LDHA of tica (Huang et al.2009).The nature of tegumental localization and with transmembrane regions suggest that LDH isoenzymes composed of subunit A or (and)B may be located in the superficial membrane of the tegument.The tegument is a syncytial structure that functions as an interface of material exchanges and interaction between parasite and host (Jones et al.2004;Van Hellemond et al.2006;Xu et al.2008).The adult T.solium inhabits the small intestine of humans,with its digestive system completely degenerated,and all nutrition including glucose intake takes place thoroughly through the tegument,rendering it a metabolically active tissue.It was reported that the activity of LDH was strong in the tegument and subtegumental muscle layers of the S.mansoni adult and sparganum (Kwak et al.1996),and Ta LDH was also immunolocalized on the tegument of the tica adult and embryonic membrane of the oncosphere (Huang et al.2009).Ts LDHA and Ts LDHB exclusively expressed in the tegument indicate the tegument is the key site of glycolysis.However,the exact topological structure and subcellular location of these two LDHs and the biochemical properties of the tetramer isozymes need furtherinvestigation.Fig.5Immunohistochemical localization of Ts LDH at the adult T.solium.The right images of a ,b ,c ,and d show the tegument and underlying tissues of the adult worm under fluorescence microscope,and the left images show the same part under optical microscope (×40).The localization of Ts LDHA and Ts LDHB is shown in the tegument by immune serum,respectively (a and c );the reddish-orange fluorescence was obviously observed as the arrows indicate.Negative control with pre-immune rat serum (b and d)Fig.4Western blot analysis of r Ts LDHA and r Ts LDHB.In a ,protein molecular weight markers (M ),r Ts LDHA and r Ts LDHB reacted with:pre-immune rat serum (a,lanes 1,2)and the corresponding anti-recombinant proteins immune serum (a,lanes 3or 4),serum from healthy swine (a,lanes 5,6),and swine infected with T.solium (a,lanes 7,8),serum from healthy people (a,lanes 9,10),and patient infected with T.solium (a,lanes 11,12);the purified r Ts LDHA reacted with the rat anti-r Ts LDHB immune serum (a,lane 13)and the purified r Ts LDHB reacted with the rat anti-r Ts LDHA immune serum (a,lane 14).In b ,protein molecular weight markers (M );the purified r Ts LDHA reacted with serum from:healthy people (b,lane 1),patient infected with T.saginata ,tica ,C.sinensis ,and S.japonicum (b,lanes 2,3,4,5);purified r Ts LDHB reacted with serum from:healthy people (b,lane 6),patient infected with T.saginata ,tica ,C.sinensis ,and S.japonicum (b,lanes 7,8,9,10)Parasitol ResComparative analysis of the sequences and structures of Ts LDHA and Ts LDHB suggested they are relatively conserved proteins with the identity of57.4%,and Western blot demonstrated a significant cross-reaction between them.This brings an important issue that of which LDH is located in the tegument or the exact constitution of isoenzymes because isozymes with different compositions of LDHA and LDHB have distinct dynamic properties and physiological function.In order to discriminate between the two subunits of LDH isoenzymes,we investigated their immunological characteristics based on the linear B-cell epitope analysis.There five linear B-cell epitopes were found in Ts LDHA;the E5is Ts LDHA specific epitope; Ts LDHB was predicted of four epitopes similar with Ts LDHA E1to E4but lacks the E5epitope.The E5 epitope will be valuable in distinguishing between Ts LDHA and Ts LDHB.The related work is being carried out.The predicted E3epitopes of Ts LDHA and Ts LDHB contain the same LDH-conserved domain at aa190–aa199, and the histidine residue located at aa193is one of the LDH active sites.Theoretically,the specific antibody against this epitope will inhibit its activity and definite the topological structure of both the LDHs in the worm.If the epitope can be bound by the antibody under the condition of intact superficial membrane of the tegument,the epitope is exposed outside of the surface of the tegument,and the antibody will play a role of inhibitor.Both r Ts LDHA and r Ts LDHB could strongly react with rat immuno-sera,swine serum,and patient serum infected with T.solium,suggesting the two recombinant proteins have immunogenicity and immunoreactivity.Obvious cross-reactions were demonstrated between T.solium and other helminths by Western blot,which is derived from the conservation of LDH from different species and indicated the Ts LDH is not a specific antigen for immunodiagnosis.In summary,this study cloned and characterized LDHA and LDHB genes from adult T.solium;Ts LDHA and Ts LDHB are not specific antigens for immunodiag-nosis,but their membrane localization in the tegument suggested they are important targets for anti-Taenia drugs and are candidate molecules for vaccine development.The linear B-cell epitope analysis of Ts LDHA and Ts LDHB discovered a Ts LDHA specific epitope,which will facilitate further studies on the functions of isoenzymes, topological structure,and relative immunological significance in vaccine research.Acknowledgments This work was supported by the National Natural Science Foundation grant(no.30760227),Guizhou Province Science-Technology Plan grant(no.2008-3060),Guizhou Province Agricultural Science Key Task Technology Projects(no.2009-3074),Guizhou Province Key Technologies R&D Program grant(no.2009-3101), Guizhou Province Governor Foundation(no.2009-82),and Guiyang City ApplicationTechnique Development grant(no.3-005).ReferencesAguilar-Diaz H,Bobes RJ,Carrero JC,Camacho-Carranza R, Cervantes C,Cevallos MA,Davila G,Rodriguez-Dorantes M, Escobedo G,Fernandez JL,Fragoso G,Gaytan P,Garciarubio A, Gonzalez VM,Gonzalez L,Jose MV,Jimenez L,Laclette JP, Landa A,Larralde C,Morales-Montor J,Morett E,Ostoa-Saloma P,Sciutto E,Santamaria RI,Soberon X,De La Torre P,Valdes V, Yanez J(2006)The genome project of Taenia solium.Parasitol Int55:127–130Alcazar O,Tiedge M,Lenzen S(2000)Importance of lactate dehydrogenase for the regulation of glycolytic flux and insulin secretion in insulin-producing cells.Biochem J352:373–380 Bern C,Garcia HH,Evans C,Gonzalez AE,Verastegui M,Tsang VC, Gilman RH(1999)Magnitude of the disease burden from neurocysticercosis in a developing country.Clin Infect Dis 29:1203–1209Bork S,Okamura M,Boonchit S,Hirata H,Yokoyama N,Igarashi I (2004)Identification of Babesia bovis L-lactate dehydrogenase asa potential chemotherapeutical target against bovine babesiosis.Mol Biochem Parasitol l36:l65–l72Camemn A,Read J,Tranter R,Winter VJ,Sessions RB,Brady RL, Vivas L,Easton A,KendrickH CSL,Barros D,Lavandera JL, Martin JJ,Risco F,García-Ochoa S,Gamo FJ,Sanz L,Leon L, Ruiz JR,GabarróR,Mallo A,FGdl H(2004)Identification and activity of a series of azole-based compounds with lactate dehydrogenase directed anti-malarial activity.J Biol Chem 279:31429–31439Conners R,Schambach F,Read J,Cameron A,Sessions RB,Vivas L, Easton A,Croft SL,Brady RL(2005)Mapping the binding site for gossypol-like inhibitors of Plasmodium falciparum lactate dehydrogenase.Mol Biochem Parasitol142:137–148Dando C,Schroeder ER,Hunsaker LA,Deck LM,Royer RE,Zhou X, Parmley SF,Vander Jagt DL(2001)The kinetic properties and sensitivities to inhibitors of lactate dehydrogenases(LDH1and LDH2)from Toxoplasma gondii:comparisons with pLDH from Plasmodium falciparum.Mol Biochem Parasitol118:23–32 Garcia HH,Gonzalez AE,Evans CA,Gilman RH(2003)Taenia solium ncet362:547–556Huang C,Hu XC,Yu XB,Lu G,Liu L,Wang LX,Yu XB(2008) Subcellular and immunohistochemistrical localization of Clonorchis sinensis lactate dehydrogenase.Chin J Zoonoses Chinese J24(10):941–944Huang J,Huang Y,Wu X,Du WY,Yu XB,Hu XC(2009) Identification,expression,characterization,and immunolocalization of lactate dehydrogenase from Taenia asiatica.Parasitol Res 104:287–293Imagawa T,Yamamoto E,Sawada M,Okamoto M,Uehara M(2006) Expression of lactate dehydrogenase-A and-B messenger ribonucleic acids in chick glycogen body.Poult Sci85:1232–1238Jones MK,Gobert GN,Zhang L,Sunderland P,McManus DP(2004) The cytoskeleton and motor proteins of human schistosomes and their roles in surface maintenance and host-parasite interactions.Bioessays26:752–765Kwak KH,Cheon EW,Kim CH(1996)Localization and characteristics of lactate and malate dehydrogenase in the sparganum and adult worm of Spirometra erinacei.Korean J Parasitol34(1):59–68 Laughton JD,Bittar P,Charnay Y,Pellerin L,Kovari E,Magistretti PJ, Bouras C(2007)Metabolic compartmentalization in the human cortex and hippocampus:evidence for a cell-and region-specific localization of lactate dehydrogenase5and pyruvate dehydrogenase.BMC Neurosci8:35Lu G,Hu XC,Peng ZY,Xie HY,Li YW,Wu ZD,Yu XB(2006) Expression and characterization of lactate dehydrogenase from Schistosoma japonicum.Parasitol Res99(5):593–596Parasitol Res。

生化英文词汇

Biochemistry> 冈崎片段oncogene ————癌基因，原癌基因one carbon unit ————一碳单位operator ————操纵基因operon ————操纵子orotic acid ————乳清酸ossification ————成骨作用oxaloacetic acid ————草酰乙酸oxidases ————氧化酶类oxidative phosphorylation ————氧化磷酸化oxidoreductase ————氧化还原酶palindrome ————回文结构pancreatic lipase ————胰脂肪酶pantothenic acid ————遍多酸pentose ————戊糖pentose phosphate pathway ————磷酸戊糖途径pepsin ————胃蛋白酶pepsinogen ————胃蛋白酶原peptide ————肽peptide bond ————肽键peptidyl site ————肽基位或P位peroxidase ————过氧化物酶phenylalanine ————苯丙氨酸phosphatidic acid ————磷脂酸phosphogluconate ————磷酸葡萄糖酸phospholipase ————磷脂酶plasmid ————质粒polycistron ————多作用子polypeptide ————多肽porphyrin ————卟啉precipitation ————沉淀preproalbumin ————前清蛋白原primary structure ————一级结构primase ————引发酶primer ————引物glucogenic amino acid ————生糖氨基酸glucokinase ————葡萄糖激酶gluconeogenesis ————糖(原)异生作用glutamic acid ————谷氨酸glutaminase ————谷氨酰胺酶glutamine ————谷氨酰胺glutathione ————谷胱甘肽glycerol ————甘油glycine ————甘氨酸glycogen ————糖原glycogen phosphorylase ————糖原磷酸化酶glycogen synthase ————糖原合成酶glycolysis ————糖酵解guanosine ————鸟苷helicase ————解链酶(解旋酶)heme ————血红素heteroduplex ————杂化双链hexokinase ————己糖激酶histamine ————组胺histidine ————组氨酸housekeeping gene ————管家基因hybridization ————杂交hydrogen bond ————氢键hydrolase ————水解酶类hydroperoxidases ————氢过氧化酶类hydrophobic bond (hydrophobic interaction) ————疏水键hydroxyapatite ————羟磷灰石hydroxymethylglutaryl CoA cleavage enzyme ————HMG CoA裂解酶hydroxymethylglutaryl CoA synthetase ————HMG CoA合酶Hydroxyproline ————羟脯氨酸acceptor site ————受位acetone ————丙酮activator ————激活蛋白，激活剂，活化物adenine (A) ————腺嘌呤adenosine ————腺苷aerobic dehydrogenase ————需氧脱氢酶alanine ————丙氨酸albumin ————白蛋白，清蛋白allopurinol ————别嘌呤醇allosteric effect ————别构(位)效应allosteric enzyme ————变构酶，别位酶allosteric regulation ————别构调节amine ————胺aminoacyl site ————A位，氨酰基位anticodon ————反密码子arginine ————精氨酸ascorbic acid ————抗坏血酸(维生素C)asparagine ————天冬酰胺aspartic acid ————天冬氨酸asymmetric transcription ————不对称转录attenuator ————衰减子base ————碱基base pairing ————碱基配对bile pigment ————胆色素biotin ————生物素biotransformation ————生物转化calcitriol ————１，２５二羟胆骨化醇（钙三醇）calcium dependent protein kinase ————Ca依赖性蛋白激酶，蛋白激酶C(C激酶) Calmodulin <生物化学Biochemistry>carbohydrate ————糖carnitine ————肉毒碱catalase ————触酶，过氧化氢酶cephalin ————脑磷脂de novo synthesis ————从头合成degradation ————降解denaturation ————变性deoxycholic acid ————脱氧胆酸deoxyribonucleotide ————脱氧核糖核苷酸dialysis ————透析dihydroxyacetone phosphate ————磷酸二羟丙酮disulfide bond ————二硫键DNA polymerase ————DNA聚合酶domain ————域,结构域,功能区donor site ————给位double helix ————双螺旋effector ————效应器,效应物elongation ————延长endopeptidase ————内肽酶enhancer ————增强子enolphosphopyruvate ————磷酸烯醇式丙酮酸enzyme ————酶essential amino acid ————必需氨基酸essential fatty acid ————必需脂肪酸exon ————外显子exopeptidase ————外肽酶fat ————脂肪feedback inhibition ————反馈抑制作用feritin ————铁蛋白ferrochelatase ————亚铁螯合酶folic acid ————叶酸free fatty acid ————游离脂肪酸free radicals ————自由基fructose diphosphatase ————果糖二磷酸酶gene cloning ————基因克隆gene expression ————基因表达gene library ————基因文库gene transfer ————基因导入,转基因genetic code ————遗传密码genetic engineering ————基因工程genetic recombination ————基因重组genome ————染色体基因，基因组globin ————珠蛋白hypocalcemia ————低钙血症induction ————诱导initiator codon ————起动信号,起始密码子intermediary metabolism ————中间代谢ionic bond ————离子键isocitrate dehydrogenase ————异柠檬酸脱氢酶isoleuc ine ————异亮氨酸isomerase ————异构酶类isozyme ————同工酶jaundice ————黄疸ketogenic amino acid ————生酮氨基酸key enzyme ————关键酶kinase ————激酶lactate ————乳酸盐lecithin ————卵磷脂leucine ————亮氨酸ligase ————连接酶linoleate ————亚油酸linolenate ————亚麻酸lipoic acid ————硫辛酸lipoid ————类脂lipoprotein ————脂蛋白lithocholic acid ————石胆酸lyases ————裂合酶类malate ————苹果酸malate aspartate shuttle ————苹果酸天冬氨酸穿梭metabolic regulation ————代谢调节mitogen activated protein kinase ————分裂原活化蛋白激酶mixed function oxidase ————混合功能氧化酶molecular cloning ————分子克隆molecular disease ————分子病monooxygenase ————单加氧酶monooxygenase system ————单加氧酶体系nicotinamide ————烟酰胺，尼克酰胺nitrogen balance ————氮平衡pyruvate carboxylase ————丙酮酸羧化酶pyruvate dehydrogenase complex ————丙酮酸脱氢酶复合体pyruvate kinase ————丙酮酸激酶quaternary structure ————四级结构recombinant DNA————重组DNAgenetic engineering ————基因工程regulatory gene ————调节基因renaturation ————复性repair ————修复replication ————复制repression ————阻遏residue ————残基respiratory chain ————呼吸链restriction endonuclease ————限制性内切核酸酶retinol ————视黄醇(维生素A)reverse transcriptase ————逆转录酶reverse transcription ————逆转录作用salting out ————盐析salvage pathway ————补救(重新利用)途径screening ————筛选secondary structure ————二级结构semiconservative replication ————半保留复制sense strand ————有意义链sequence ————序列serine ————丝氨酸signal recognition particle ————信号肽识别颗粒silencer ————抑制子simple protein ————单纯蛋白质specificity ————特异性splicing ————剪接作用squalene ————鲨烯stage specificity ————阶段特异性stercobilinogen ————粪胆素原stress ————应激structural gene ————结构基因substrate ————作用物substrate level phosphorylation ————作用物(底物)水平磷酸化subunit ————亚单位，亚基succinate dehydrogenase ————琥珀酸脱氢酶supersecondary structure ————超二级结构Taurine ————牛磺酸telomerase ————端粒酶telomere ————端区(端粒)template strand ————模板链termination ————终止terminator ————终止子terminator codon ————终止信号tertiary structure ————三级结构thiamine ————硫胺素(维生素B1) threonine ————苏氨酸thymidine ————胸苷，胸腺嘧啶核苷thymine (T) ————胸腺嘧啶tocopherol ————生育酚proalbumin ————清蛋白原processing ————加工proenzyme ————酶原proline ————脯氨酸promoter ————启动基因(启动子)，催化剂prosthetic group ————辅基protease ————蛋白酶pyridoxal ————吡哆醛pyridoxamine ————吡哆胺。

分子克隆技术

大肠杆菌转化体系的建立：感受态菌制备经氯化钙处理的大肠杆菌能够摄取质粒 DNA。 1.双抗性（TerKar)重组质粒DNA及双抗性转化大肠杆菌细胞的获得。
2.真核动物非洲爪蟾的基因在原核细胞中转录
意义：1.解决了无复制能力的DNA片断在宿主细胞中进行繁殖克隆的关键问题；2.质粒分子可作为基因克隆的载体将外源DNA分子导入宿主细胞；3.真核细胞的基因可被转移到原核细胞中进行克隆及表达。
分子克隆技术
分子克隆（molecular cloning）
又称为基因克隆、基因工程、DNA克隆、重组DNA技术
克隆（Clone）指通过无性繁殖过程所产生的与亲代完全相同的子代群体。
分子克隆指按照人的意愿，在体外将某种生物的DNA片断插入到载体（质粒、病毒等）中，形成遗传物质的新组合---重组DNA分子（重组子），然后将重组子转移到宿主细胞中进行复制或表达，即对DNA分子进行克隆，以获得该DNA分子的大量拷贝。
基本原理：将编码某一多肽或蛋白质的基因（外源基因）组装到细菌质粒（质粒是细菌染色体外的双链环状 DNA分子）中，再将重组质粒转入大肠杆菌，这样重组质粒就随大肠杆菌的增殖而复制，从而克隆基因或表达出外源基因编码的相应多肽或蛋白质。
At Stanford Berg and his colleagues spliced two DNA molecules together in vitro and were able to insert a set of three genes responsible for metabolizing galactose in Escherichia coli into the SV40 DNA genome.

生物基因的实验报告(3篇)

第1篇一、实验名称生物基因的提取与鉴定二、实验目的1. 学习生物基因提取的方法和原理。

2. 掌握DNA提取、纯化及检测的基本操作。

3. 鉴定提取的DNA是否为纯DNA。

三、实验原理生物基因提取的原理是利用细胞破碎、蛋白质变性、DNA与蛋白质分离等方法，从生物细胞中提取出DNA。

实验中常用的提取方法有酚-氯仿法、柱层析法等。

DNA鉴定通常采用琼脂糖凝胶电泳和紫外分光光度法。

四、实验材料1. 植物材料：新鲜植物叶片（如水稻、玉米等）。

2. 试剂：酚-氯仿、NaCl溶液、Tris-HCl缓冲液、琼脂糖、DNA标准品、DNA酶、DNA荧光染料等。

3. 仪器：离心机、电泳仪、紫外分光光度计、显微镜等。

五、实验方法1. 植物材料的处理：将新鲜植物叶片洗净，剪成小块，加入适量液氮研磨成粉末。

2. DNA提取：（1）将研磨好的植物粉末加入酚-氯仿溶液，充分振荡，静置分层。

（2）取上清液，加入等体积的NaCl溶液，混匀后加入等体积的冷无水乙醇，充分混匀。

（3）室温静置30分钟，离心取沉淀。

（4）将沉淀溶于适量Tris-HCl缓冲液，得到粗提DNA。

3. DNA纯化：（1）将粗提DNA加入柱层析柱，用适量Tris-HCl缓冲液洗脱。

（2）收集洗脱液，加入适量无水乙醇，静置沉淀。

（3）离心取沉淀，溶于适量Tris-HCl缓冲液，得到纯化DNA。

4. DNA鉴定：（1）取适量纯化DNA，用紫外分光光度计测定其浓度。

（2）将纯化DNA与DNA标准品在同一琼脂糖凝胶电泳槽中电泳，观察DNA条带。

（3）对电泳后的凝胶进行紫外透射，观察DNA条带。

六、实验结果1. DNA浓度：通过紫外分光光度计测定，纯化DNA浓度为20ng/μl。

2. 琼脂糖凝胶电泳：纯化DNA在琼脂糖凝胶电泳中呈现清晰的条带，与DNA标准品对照，条带大小相符。

七、实验讨论1. 在实验过程中，注意避免DNA污染，如使用无DNA酶的试剂、操作过程严格无菌等。

2. 提取的DNA浓度受植物材料、提取方法等因素影响，可通过优化实验条件提高DNA浓度。

Molecular Cloning_Technical guide

Visit
22–23 Vector and Insert Joining
22–23 DNA Ligation 22 Protocol 22 Tips for Optimization 23 Product Selection
24 Transformation
5 Recombinational Cloning 8 cDNA Synthesis 9 Restriction Enzyme Digestion 9 Protocol 9 Tips for Optimization 10–15 Performance Chart 16–17 PCR 16 Protocol 16 Tips for Optimization 17 Product Selection
Molecular Cloning
Technical Guide
Overview
table of contents
3–5 Cloning Workflow Descriptions
Molecular Cloning Overview
Molecular cloning refers to the process by which recombinant DNA molecules are produced and transformed into a host organism, where they are replicated. A molecular cloning reaction is usually comprised of two components: 1. The DNA fragment of interest to be replicated 2. A vector/plasmid backbone that contains all the components for replication in the host DNA of interest, such as a gene, regulatory element(s), operon, etc., is prepared for cloning by either excising it out of the source DNA using restriction enzymes, copying it using PCR, or assembling it from individual oligonucleotides. At the same time, a plasmid vector is prepared in a linear form using restriction enzymes (REs) or Polymerase Chain Reaction (PCR). The plasmid is a small, circular piece of DNA that is replicated within the host and exists separately from the host’s chromosomal or genomic DNA. By physically joining the DNA of interest to the plasmid vector through phosphodiester bonds, the DNA of interest becomes part of the new recombinant plasmid and is replicated by the host. Plasmid vectors allow the DNA of interest to be copied easily in large amounts, and often provide the necessary control elements to be used to direct transcription and translation of the cloned DNA. As such, they have become the workhorse for many molecular methods such as protein expression, gene expression studies, and functional analysis of biomolecules. During the cloning process, the ends of the DNA of interest and the vector have to be modified to make them compatible for joining through the action of a DNA ligase, recombinase, or an in vivo DNA repair mechanism. These steps typically utilize enzymes such as nucleases, phosphatases, kinases and/or ligases. Many cloning methodologies and, more recently kits have been developed to simplify and standardize these processes. This technical guide will clarify the differences between the various cloning methods, identify NEB® products available for each method, and provide expert-tested protocols and FAQs to help you troubleshoot your experiments.

基因工程复习总结

1.定义基因工程（gene engineering),又称DNA重组技术(recombinant DNA technique)、分子克隆（molecular cloning)和遗传工程（genetic engineering),是上世纪70年代兴起的技术科学.通过特殊的酶处理，使遗传物质在体外发生重组，从而产生自然界从未有过的重组DNA分子（至少包含两种不同生物的DNA片段）。

在他们进入一定的生物寄主后，不仅可以得到维持，而且可以得到扩增，其上的外源基因甚至可以得到表达。

2．孟德尔遗传定律的重新发现者:荷兰的德弗里斯(H. De Vries )德国的科伦斯(C. Correns).奥地利的契马克(E. Seysenegg-Tschermak)3.外源DNA进入细菌后，面临两种命运：一是被限制，即被降解；二是被修饰，即发生甲基化，不被降解。

4 . 原核细胞中限制和修饰系统I类酶酶分子：三亚基双功能；识别位点：二分非对称序列；切割位点：距识别位点1000bp ；限制性反应与甲基化反应：互斥；限制作用需要A TP: 需要II类酶酶分子：内切酶与甲基化酶分离; 识别位点：4-6bp序列，回文结构; 切割位点：在识别位点中或靠识别位点; 限制性反应与甲基化反应：分开反应; 限制作用需要A TP: 需要III类酶酶分子：二亚基双能; 识别位点：5-7bp非对称序列; 切割位点：在识别位点下游24-26bp; 限制性反应与甲基化反应：同时竟争; 限制作用需要A TP: 不需要5. 命名规则：生物体属名的第一大写字母和种名前两个小写构成基本名称基本名称+ 菌株名的字母+ 罗马字母（发现顺序）Hind IIIHaemophilus infuenzae d株中的第三个酶EcoR I基因位于Escherichia coli 抗药性R质粒上E co R I细菌属名细菌种名菌株类型有几种限制酶6.识别的序列一般为4-8bp，常见的为6bpEg:5bp识别位点Eco R II CCWGG W表示A或T；S表示C或G7回文结构：DNA局部双螺旋以某一对称轴旋转180度后，与另一侧的互补片段的顺序完全的DNA结构8. M表示A或C；K表示G或T; Y表示C或T；R表示A或G9切割的位置，有的在内部，有的在外部，外部的又有两端、两侧和单侧之分。

基因克隆

RNA提取两要素
• 忌讳贪多忌讳贪多：不能指望1ml Trizol 提取100mg以上组织，一般50mg用1mlTrizol较合适 • 速战速决速战速决：尽量缩短提取时间，不要完全照搬 Protocol,比如匀浆后如果没有很多组织碎片的话，可以立即加入氯仿，立即离心，14000rpm离心 10min,取完上清加入0.6~1体积的异丙醇，立即高速离心（14,000rpm,5min），..... 总之尽量快速！
• 扩增反应条件 • 94℃预变性3min；然后进行30个循环反应，其温度循环条件为：94℃ • 变性30s,50℃ 30s，72℃延伸1min；循环结束后72℃再延伸10min。
PCR的基本反应的基本反应变性
95˚C
延伸 72˚C
退火
Tm-55˚C
P1 第一次循环
94℃ ℃
Taq DNApol. P2
E1 E3 E2 X E4 E5 E5 E1
B E4 E3
E2
3、琼脂糖凝胶电泳（1）原理：电荷效应和分子筛效应原理：移动方向：负极到正极（2）DNA移动方向：负极到正极移动方向（3）迁移速率：DNA分子的大小和构型）迁移速率：分子的大小和构型与相对分子量的对数值成反比相对分子量相同时超螺旋状DNA 线形DNA 带缺刻的环状DNA 超螺旋状DNA > 线形DNA > 带缺刻的环状DNA
2、限制性内切酶（restriction endonuclease）、限制性内切酶（）细菌的限制修饰系统（restriction-modification system））（1）类型：）类型： type I、type III 、限制性内切活性切点远离识别位点（移动需要ATP）切点远离识别位点（移动需要） type II 限制性内切活性（限制性内切活性（Mg++、Na+、pH、37°C）、 ° ）切点与识别位点基本一致

分子克隆

概述
• 分子克隆操作的主要对象是基因。 • 待研究或检测的特定基因被称为目的基因。
• 目的基因通过相应的基因载体导入宿主细胞，并借助宿主细胞体系进行复制或表达，所以对宿主细胞DNA而言，目的基因被称为外源基因或外源DNA。
分子克隆的目的
• 分子克隆的目的就是研究基因与改造基因。 • 根据研究目的的不同，研究对象有所同，目的基因可大致分为三类：病毒基因、原核基因及真核基因。 • 病毒基因组与原核生物基因组相对简单，较易获得目的基因； • 而真核生物基因组庞大复杂，要从众多的基因中获得所需的目的基因难度相对较大。
限制性核酸酶内切酶活性及其影响因素
• 限制性核酸酶内切酶活性大小体现在酶对DNA的水解程度不同，常用酶的活性单位来衡量其活性大小。 • 酶的活性单位是指1微克纯DNA在指定缓冲液中，于37℃孵育1h完全酶解DNA中所以同一限制性内切酶位点所需要的酶。 • 影响限制酶反应的因素很多，DNA制品中的污染物（如蛋白质、酚、氯仿、乙醇、EDTA、SDS、高盐浓度）及缓冲条件等，均能抑制酶的活性。
限制性核酸内切酶
• 通过切割相邻的两个核苷酸残基之间的磷酸二酯键，导致核酸分子多核苷酸链水解断裂的酶叫做核酸酶。
核糖核酸酶核酸酶脱氧核糖核酸酶
• 核糖核酸酶：专门水解RNA的核酸酶 • 脱氧核糖核酸酶：专门水解DNA分子的核酸酶 • 核酸按水解核酸分子的不同方式，又可分为两种类型： • 核酸外切酶是从核酸分子的末端开始，逐个降解核苷酸。 • 核酸内切酶：从核酸分子的内部水解磷酸二酯键，使之断裂成小片段。
克隆的基本原理
• 将编码某一多肽或蛋白质的基因（外源基因）组装到细菌质粒（质粒是细菌染色体外的双链环状 DNA分子）中，再将这种质粒（重组质粒）转入大肠杆菌体内，这样重组质粒就随大肠杆菌的增殖而复制，从而表达出外源基因编码的相应多肽或蛋白质。