人卵巢癌cDNA表达文库的构建

Construction of cDNA Library of Ovarian Carcinoma

以polyA^+ mRNA提取试剂盒从卵巢癌组织中获得高质量的polyA^+ mRNA,并以此合成第1、2链cDNA.cDNA两端补平后加EcoR Ⅰ接头,Xbo酶切,通过分级分离去除<400bP的片段.取100ng cDNA与噬菌体表达载体Uni-ZAP XR连接,体外进行噬菌体DNA包装,并立即进行文库的滴定和扩增,获得卵巢癌cDNA表达文库.该文库原始重组子为10~6独立克隆;重组率>99%;并以引物PUCⅠ、PUCⅡ扩增插入片段,平均大小约为1.1kb.证实此cDNA表达文库合格.以PCR从此cDNA表达文库中筛选HLA-DPB和β-actin基因,获得成功.

We isolated polyA^+ mRNA direcdy from tumor tissue of ovarian carcinoma using Oligotex^(TM) Direct mRNA Kit (QIAGEN) to synthesize the first and second strand cDNA. The ds-cDNA termini were blunted with pfu DNA poly-rnerase. The blunted cDNAs were added EcoR I adaptor, and then were digested by Xho I . Small cDNA molecules( < 400bp) were removed through size fraction. After ligating the cDNAs into expression vector of Uni-ZAP XR, the ligated products were packaged in vitro and the bacterophage particles infected the host strains XLl-blue mrf' . The cDNA expression library was screened for the genes of HLA-DPB and βactin with PCR. The ds-cDNAs were confirmed to be more than 400bp in size. The cDNA expression library of ovarian carcinoma was showed 10~6 recombinant plaques. The recombination rate was more than 99% . The average size of the inserts was 1. 1kb. The PCR amplified genes of HLA-DPB and βactin, 327bp and 662bp respectively were successfully obtained from the library. All data showed that the cDNA expression library we constructed was of high quality and could be used for further immunoscreening.