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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0.4 kb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0.95 kb,长度在1.0~2.0 kb的4个,0.75~1.0 kb的4个,0.5~0.75 kb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 construct cDNA library of human primary hepatocellular carcinoma (HCC) and screen the genes specifically related to the occurrence of primary hepatocellular carcinoma (HCC), so as to lay a foundation for exploring the pathogenesis and gene therapy of primary HCC. Methods The total RNA was extracted from human primary hepatocellular carcinoma tissue and purified. The single-stranded cDNA was reverse transcribed and the double-stranded cDNA was synthesized by long-distance polymerase chain reaction (PCR). After protease K hydrolysis and purification, the Sfi Ⅰ enzyme Cut; the enzyme-digested products were fractionated, the components over 0.4 kb were recovered and ligated with the λTriplEx2 vector; the attached products were packaged in vitro to produce an unexpanded library; the titer and recombination efficiency of the unamplified library were examined , Amplifying the library; detecting the titer and recombination efficiency of the amplified library, randomly selecting 14 plaques and performing PCR amplification with common primers at both ends of the vector cloning site to detect the quality of the constructed cDNA library . Results The titer of the untreated library was 1.37 × 106pfu / ml and the efficiency of recombination was 97.46%. The amplified library titer was 1.28 × 109pfu / ml and the recombination efficiency was 99.06%. The average length of the inserted fragment was 0.95 kb, Four from 1.0 to 2.0 kb, four from 0.75 to 1.0 kb, and six from 0.5 to 0.75 kb. Conclusion A human primary hepatocellular carcinoma cDNA library has been constructed successfully on the basis of hepatitis B cirrhosis.