人原发性肝癌组织cDNA文库的构建及鉴定

Construction and identification of a cDNA library from human liver tissues of primary liver carcinoma

目的构建人原发性肝癌组织cDNA文库,筛查与原发性肝癌发生特异相关的基因,为探讨原发性肝癌的发病机制及基因治疗奠定基础.方法提取人原发性肝癌组织总RNA并纯化其mRNA;逆转录合成单链cDNA,长距离聚合酶链反应(PCR)合成双链cDNA;PCR产物经蛋白酶K水解并纯化后,进行SfiⅠ酶酶切;将酶切产物进行分级分离,回收0.4kb以上的组分,并与λTriplEx2载体连接;将连接产物经体外蛋白包装,产生未扩增文库;检测未扩增文库的滴度和重组效率后,进行文库扩增;检测扩增后文库的滴度和重组效率,随机挑取14个噬菌斑,用载体克隆位点两端的通用引物进行PCR扩增,以检测所构建的cDNA文库的质量.结果未扩增文库的滴度为1.37×106pfu/ml,重组效率为97.46%;扩增后文库滴度为1.28×109pfu/ml,重组效率为99.06%;插入片段平均长度为0.95kb,长度在1.0～2.0kb的4个,0.75～1.0kb的4个,0.5～0.75kb的6个.结论已成功构建一个乙型肝炎肝硬化基础上发生的人原发性肝癌组织cDNA文库.

Objective To screen genes associated with the occurrence of hepatocarcinoma, a cDNA library of human liver tissue of primary liver carcinoma was constructed. Methods Total RNA was extracted and mRNA was purified, mRNA was reversed to first-strand cDNA which was amplified to double-strand cDNA by long distance PCR. PCR products were digested by proteinase K and Sfi Ⅰ , and were fractionated by CHROMA SPIN-400 column. The cDNA of length longer than 0.4 kb were collected and ligated to λTriplEx2 vector. The λ-phage packaging reaction for the ligated DNA was performed to produce an unamplified library. Thereafter, the unamplified library was titered and the percentage of recombinant clones were detected. In the end, fourteen plaques were randomly selected and amplified by PCR using universal primers from vector in order to test the quality of the obtained library. Results The titers of unamplifed and amplified libraries were 1.37 × 10^6 pfu/ml and 1.28 × 10^9 pfu/ml respectively. The percentage of recombinants of them were 97.46% and 99.06% respectively. The average length of the inserts was 0.95 kb. Conclusions A high quality cDNA library from human liver tissue of primary liver carcinoma was constructed successfully, and it lays solid foundation not only for screening and cloning new special genes associated with the occurrence of hepatocarcinoma, but also for gene therapy of it.