大肠杆菌cDNA文库的构建与质量分析  

Construction of a cDNA Library of Escherichia Coli and Its Quality Analysis

作　　者：肖业[1] 王婷婷[1] 向双林[1] 

机构地区：[1]湖南师范大学生命科学学院,中国长沙410081

出　　处：《湖南师范大学自然科学学报》2016年第4期29-33,共5页Journal of Natural Science of Hunan Normal University

基　　金：国家973计划资助项目(2010CB529900);长沙市科技局资助项目(K1205221-31)

摘　　要：采用Stratagene公司的cDNA文库构建试剂盒构建大肠杆菌的cDNA文库,从中筛选出与RNaseⅢ有相互作用的蛋白,为研究RNaseⅢ蛋白在原核生物中的作用奠定基础.用Trizol试剂提取大肠杆菌的总RNA,在E.coli poly（A）Polymerase作用下给mRNA特异性地加上poly（A）尾,分离纯化mRNA,利用mRNA作为模板反转录合成双链DNA.经pfx酶补平双链末端,在两端加上EcoRⅠ接头,XhoⅠ酶切消化产生粘性末端.用CHROMA SPIN-400column分级分离纯化cDNA片段,与p TRG XR载体连接后,电击转入XL1-BLUE MRF＇菌电转感受态中.得到原始文库,经计算文库的容量达到3×10^6个克隆.用PCR方法测得文库的重组率为100%,插入片段的平均长度基本位于300 bp以上,测定放大文库的滴度为：3.15×10^10pfu/m L.说明构建的大肠杆菌cDNA文库质量比较高,适合用于筛选目的基因克隆.To investigate the role of RNaseⅢin prokaryote, a cDNA library of Escherichia coli was construc-ted using the cDNA library construction kit purchased from Stratagene, which could be further used for identifying RNase Ⅲinteracting-proteins. In this study, the cDNA library of E. coli was constructed using the following meth-od：the total RNA of E. coli was extracted using Trizol kit, and the poly（ A） tail was specifically added to mRNA with E. coli poly（ A） Polymerase. After that, mRNA was separated, purified, and reverse-transcripted into double-strand DNA. The ds-cDNA termini was blunted with pfu DNA polymerase. The blunted cDNAs were added to EcoRⅠadaptors and then digested by XhoⅠ. The cDNA size was fractionated by CHROMA SPIN-400 column. These purified fragments were ligased into the pTRG XR vector and transformed into XL1-BLUE MRF′ competent cells, which generated the oriental unamplified cDNA library. The unamplified library contained about 3 × 106 recombi-nants. The PCR results showed that the percentage of positive recombinant clones was 100%. The length of the in-serted cDNA fragment was over 300 bp. The titer of the amplified library was 3. 15 × 1010 pfu/mL. The quality of the constructed E. coli library was excellent and helpful to screen new genes in the future.

关 键 词：大肠杆菌 细菌双杂交 CDNA文库 

分 类 号：Q78[生物学—分子生物学]