cDNA文库及其在原虫研究中的应用

cDNA文库构建和筛选是基因克隆的重要方法之一,它是目前发现新基因和研究基因功能的基本工具.从cDNA文库中可以筛选到目的基因,并直接用于该基因的表达.由于cDNA文库在基因分离和克隆中具有重要作用,因此其应用也日益广泛.简要介绍自cDNA文库创建以来,发展起来的各类文库及其构建cDNA文库的方法.作者重点阐述了弓形虫、利什曼原虫、阴道毛滴虫、疟原虫等原虫cDNA文库的构建及其应用.     

cDNA文库构建方法的进展

cDNA文库构建是基因克隆的重要方法之一。从cDNA文库中能够筛选到所需的目的基因,并直接用于该目的基因的表达,它是发现新基因和研究基因功能的基础工具,本文介绍几种cDNA文库构建中的改进技术,并着重阐述其原理和特点。     

大乳头水螅RACE cDNA文库的构建

目的:为筛选和克隆大乳头水螅发育调控相关基因的全长cDNA,构建大乳头水螅RACE cDNA文库.方法:提取大乳头水螅总RNA后从其中分离mRNA,运用SMART技术构建RACE cDNA文库.为鉴定所构建文库的质量,根据GenBank中大乳头水螅actin基因cDNA序列设计5'RACE和3'RACE的引物及用于扩增actin基因编码区全长序列的引物.结果:琼脂糖凝胶电泳结果表明,RACE cDNA文库中全长cDNA的长度集中在500-2 000bp之间.5'RACE、3'RACE PCR及扩增actin基因编码区全长序列时均以本文构建的大乳头水螅RACE cDNA文库为模板,这3个PCR反应均能扩增出产物,产物大小与目标片段预计大小相似.PCR产物分别经T/A克隆及测序后证明为大乳头水螅actin基因cDNA的相应序列.结论:RACE cDNA文库的成功构建为通过RACE方法获得大乳头水螅功能基因cDNA全长序列奠定了基础.     

烤烟品种南江3号均一化全长cDNA文库构建

目的:为了获得功能基因的信息,构建烤烟品种南江3号的均一化cDNA文库.方法:用RNeasy Plant Mini Kit提取烤烟南江3号叶片和花的RNA,用反转录酶逆转录合成第一链eDNA.以LD-PCR扩增获得的双链cDNA为模板,采用基于双链特异性核酸酶(Duplex-Specific Nuclease,DSN)的均一化cDNA文库技术,构建南江3号盛花期的均一化cDNA文库.结果:构建了南江3号盛花期的均一化cDNA文库,文库重组率大为97.47%,库容量约为1.26×10<'6>,插入片段平均长度大于1.2kb.从文库中随机48个克隆进行PCR检测,挑取20个克隆进行测序,序列通过BLAST比对结果显示文库可能包含大量基因和ESTs序列.结论:该文库为研究南江3号的基因功能和资源提供材料来源.     

正常人肝细胞cDNA文库的构建及应用分析

目的利用Gubler-Hoffman法构建了正常人肝细胞的cDNA文库以筛选肝细胞内部与乙肝病毒感染相关的基因。方法首先采用TRIzol法提取正常人肝细胞总RNA,纯化mRNA。逆转录合成单链cDNA,然后合成双链cDNA。用Spin Column回收0.4kb以上片段,然后与Vector pAP3neo进行连接,利用电刺激转化法导入E.coliDH10B,利用PCR法检测文库的重组效率。结果扩增后的文库重组率为93.3%。结论已经成功地构建了正常人肝组织的cDNA文库,该文库可用于筛选与乙肝相关的基因及用于基因芯片的制作。     

鼻咽癌组织cDNA文库的构建及抗原基因的筛选

构建人鼻咽癌组织cDNA文库,以SEREX方法从cDNA文库中筛选鼻咽癌抗原基因。采用确诊鼻咽癌患者新鲜活检癌组织构建cDNA文库,测定原始文库滴度,进行蓝白筛选以确定文库的重组率。以建库组织来源患者的自身血清,采用“一对一” 的血清学方法筛选所构建的cDNA文库,阳性克隆经PCR检测鉴定后进行序列分析。经测定原始文库滴度为7.28×10⁶pfu/mL,含3.64×10⁶个重组子,重组率为94%,扩增文库滴度为3.8×10⁹pfu/mL,cDNA插入片段大小在0.5～3.0kb之间。文库经三轮血清学筛选共获得23个阳性克隆,分别代表了16个独立的cDNA插入片段（抗原基因）。其中10个与已知基因高度同源,另外6个基因与GenBank中已知基因的部分同源,其中有3个是新基因。利用SMART技术构建了高质量的人鼻咽癌组织cDNA表达文库,有利于以cDNA文库为基础的进一步的实验研究。应用SEREX技术初步筛选鼻咽癌组织cDNA文库,共得到16个鼻咽癌相关抗原基因,其中有3个是新基因,可能为鼻咽癌的免疫学研究提供新的研究分子。     

水稻穗发育相关基因酵母双杂交cDNA文库的构建

利用cDNA文库筛选与已知功能蛋白质互作蛋白质,是研究蛋白质之间相互作用的重要途径之一。本试验利用水稻武运粳7号(95-22)穗为材料,构建了一个穗发育相关基因酵母双杂交cDNA文库,试验表明,本实验构建的cDNA初级文库容量和目的文库容量分别为1.44×107 CFU/mL和9.6×106 CFU/mL,重组率均为95%;初级文库插入片段长度介于500～2 000 bp之间,目的文库插入片段长度介于1 000～2 000 bp之间,两者平均插入片段长度均在1 000 bp以上;通过NCBI数据库、RicexPro数据库和Rice Genome Annotiation Project数据库对目的文库中的19个单克隆测序分析得知,19个克隆测序序列匹配17个相应的基因,文库重复率为10.5%,其中16个基因均在穗部有表达。以上试验表明,本实验构建的cDNA文库是一个质量较高的文库,可以直接用于穗发育相关基因筛选。     

芜菁消减文库特异基因片段功能的初步分析

目的：对4个消减cDNA文库中筛选到的特异基因片段进行功能分析,为筛选津田芜菁和赤丸芜菁花青素合成的特异基因和代谢途径奠定基础。方法：以不同处理的津田芜菁和赤丸芜菁块根为材料,采用抑制削减杂交构建4个消减cDNA文库并富集特异基因群体,同时对消减文库的特异基因片段进行初步的生物信息学分析。结果：通过功能聚类、代谢途径分析和基因注释等手段分析了消减cDNA文库的特异基因片段。结论：对消减文库特异基因片段的功能分析,为进一步分离和鉴定依光型和非依光型花青素合成相关基因奠定了基础。     

基因预测与PCR技术相结合从cDNA文库中获取表达基因的研究

应用计算机工具、GenScan软件预测中国美利奴绵羊MHC Class I区段的BAC文库中453oⅡ克隆的基因数目、特性及结构,建立一种可以从cDNA文库中简便有效获取表达基因的技术方法。选取4个预测基因作研究对象设计引物,应用PCR技术,对已构建好的cDNA文库进行PCR扩增,回收"目的基因"片段并连接pGEM-T载体,转DH5α大肠杆菌中扩增后测序。琼脂糖凝胶电泳检测PCR扩增产物,cDNA文库中有目的条带,测序结果与GenBank进行Blast分析,分析结果表明这些基因与羊的基因均具有99%以上的相似性。因此应用基因预测分析与PCR结合技术可简便迅速的从cDNA文库中获取表达基因。     

SMART技术构建栀子cDNA文库

目的:构建栀子叶片cDNA文库。方法:提取栀子叶片总RNA。利用SMART技术合成双链cDNA。双链cDNA经限制酶Sfil酶切后与pDNR-LIB质粒连接。利用电刺激转化法将重组质粒导入E.coli DH5α而获得文库。利用PCR法检测文库的重组率。结果:原始文库滴度为2.63×105cfu/ml。随机检测文库中的15个克隆,表明重组率约为86.7%。选择14个插入片段的长度在400bp以上的克隆进行测序和生物信息学分析,结果预测的全长基因占所检测序列的64.3%。结论:成功构建了栀子叶片的cDNA文库,为栀子基因的结构和功能的研究提供了基础。     

几种全长cDNA文库构建方法比较

全长cDNA文库是高效、大规模获得基因全序列信息的一条有效途径,尤其是对基因组庞大,近期内尚不能进行全基因组测序的生物来说,是一条开展功能基因组研究的重要途径。本文对几种全长cDNA文库的构建方法进行了概述,针对各种方法的原理及优缺点做了分析、比较,并结合本实验室的结果,重点介绍了Cap-trapper法在小麦全长cDNA文库中的应用及文库中全长基因比例判定方法。     

Complementary DNA libraries

The generation of complete and full-length cDNA libraries for potential functional assays of specific gene sequences is essential for most molecules in biotechnology and biomedical research. The field of cDNA library generation has changed rapidly in the past 10 yr. This review presents an overview of the method available for the basic information of generating cDNA libraries, including the definition of the cDNA library, different kinds of cDNA libraries, difference between methods for cDNA library generation using conventional approaches and a novel strategy, and the quality of cDNA libraries. It is anticipated that the high-quality cDNA libraries so generated would facilitate studies involving genechips and the microarray, differential display, subtractive hybridization, gene cloning, and peptide library generation.     

Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts

The normalization and subtraction of highly expressed cDNAs from relatively large tissues before cloning dramatically enhanced the gene discovery by sequencing for the mouse full-length cDNA encyclopedia, but these methods have not been suitable for limited RNA materials. To normalize and subtract full-length cDNA libraries derived from limited quantities of total RNA, here we report a method to subtract plasmid libraries excised from size-unbiased amplified lambda phage cDNA libraries that avoids heavily biasing steps such as PCR and plasmid library amplification. The proportion of full-length cDNAs and the gene discovery rate are high, and library diversity can be validated by in silico randomization.     

SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones

Background  

cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library.     

Characterization of size-fractionated cDNA libraries generated by the in vitro recombination-assisted method.

We here modified a previously reported method for the construction of cDNA libraries by employing an in vitro recombination reaction to make it more suitable for comprehensive cDNA analysis. For the evaluation of the modified method, sets of size-selected cDNA libraries of four different mouse tissues and human brain were constructed and characterized. Clustering analysis of the 3' end sequence data of the mouse cDNA libraries indicated that each of the size-fractionated libraries was complex enough for comprehensive cDNA analysis and that the occurrence rates of unidentified cDNAs varied considerably depending on their size and on the tissue source. In addition, the end sequence data of human brain cDNAs thus generated showed that this method decreased the occurrence rates of chimeric clones by more than fivefold compared to conventional ligation-assisted methods when the cDNAs were larger than 5 kb. To further evaluate this method, we entirely sequenced 13 human unidentified cDNAs, named KIAA1990-KIAA2002, and characterized them in terms of the predicted protein sequences and their expression profiles. Taking all these results together, we here conclude that this new method for the construction of size-fractionated cDNA libraries makes it possible to analyze cDNAs efficiently and comprehensively.     

Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics

We describe the construction and characterization of two lambda surface displayed cDNA expression libraries derived from human brain and mouse embryo. cDNA inserts were obtained by tagged random-priming elongation of commercially available cDNA libraries and cloned into a novel lambda vector at the 3' end of the D capsid protein gene, which produced highly complex repertoires (1x10(8) and 2x10(7) phage). These libraries were affinity selected with a monoclonal antibody against the neural specific factor GAP-43 and with polyclonal antibodies that recognize the EMX1 and EMX2 homeoproteins. In both cases rapid identification of specific clones was achieved, which demonstrates the great potential of the lambda display system for generating affinity selectable cDNA libraries from complex genomes.     

Use of gene networks for identifying and validating drug targets

We propose a new method for identifying and validating drug targets by using gene networks, which are estimated from cDNA microarray gene expression profile data. We created novel gene disruption and drug response microarray gene expression profile data libraries for the purpose of drug target elucidation. We use two types of microarray gene expression profile data for estimating gene networks and then identifying drug targets. The estimated gene networks play an essential role in understanding drug response data and this information is unattainable from clustering methods, which are the standard for gene expression analysis. In the construction of gene networks, we use the Bayesian network model. We use an actual example from analysis of the Saccharomyces cerevisiae gene expression profile data to express a concrete strategy for the application of gene network information to drug discovery.     

Phage display in pharmaceutical biotechnology

Over the past year, methods for the construction of M13 phage-display libraries have been significantly improved and new display formats have been developed. Phage-displayed peptide libraries have been used to isolate specific ligands for numerous protein targets. New phage antibody libraries have further expanded the practical applications of the technology and phage cDNA libraries have proven useful in defining natural binding interactions. In addition, phage-display methods have been developed for the rapid determination of binding energetics at protein-protein interfaces.