人正常肾组织全长cDNA文库的构建及鉴定

Construction and identification of a cDNA library of human normal kidney tissues

目的 构建人正常肾组织的cDNA文库并鉴定文库质量。方法 运用mRNA 5′末端的模板转换方法以powerscript逆转录酶进行转录 ,在mRNA的 5′末端添加一段 5′ oligo做为延伸后的模板 ,从而富集全长cDNAs。扩增后的cDNAs经sfiⅠ酶切、柱层析洗脱 ,重组于λTripIEx2 载体并包装后 ,测定滴度、重组率 ,扩增文库。随机挑取 8个噬菌斑行PCR反应扩增插入片段。结果 构建的cDNA文库滴度为 2 .6× 10 6pfu·mL-1,重组率 >95 % ,扩增后滴度达 9× 10 11pfu·mL-1。 8个插入片段长度为 0 .7～ 2 .0kb。结论 构建的人正常肾组织cDNA文库为高效全长的cDNA文库 ,符合cDNA文库的要求 ,可以用探针、抗体等做免疫学筛选 ,进一步探寻与肾脏疾病相关的基因。

Objective To construct a full-length cDNA library of human normal kidney tissues and identify the quality of the library. Methods By using the template-switching mechanism at 5′end of mRNA technique to construct the library, a powerscript reverse transcriptase was used to transcribe, and a 5′-oligo fragment as an extended template was added to 5′ end of mRNA to enrich full-length cDNAs. After amplification, the ds cDNAs digested by sfi I and size-fractionated by columns were recombined into λTripIEx 2 vectors. After package, the recombinant vectors were titered and the recombinant rate (blue/white) was determined,then the library was amplified. We identified the library using PCR reaction to determine the size of the inserts. Results The titer of cDNA library was 2.6×10 6pfu·mL -1, the rate of recombinant was above 95%, and the titer of amplified library was 9×10 11pfu·mL -1. The insert size ranged from 0.7 to 2 kb. Conclusion The cDNA library of human normal kidne we constructed is a highly efficient one and can be used for screening by probe and antibody to find the genes related to kidney diseases.