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目的快速构建混合人原发型肝癌组织cDNA文库。方法从混合人原发型肝癌组织分离总RNA,以含有sfiIB酶切位点的oligo(dT)引物合成第1链cDNA,以含sfiIA酶切位点的SMART寡核苷酸为引物经LD-PCR扩增双链cDNA,双链cDNA经sfiI(IA&IB)酶切,以CHROMA SPIN-400柱分级分离cD-NA,收集符合需要的cDNA片段并纯化,随后将之与λTriplEx2载体连接经体外包装成噬菌体cDNA文库。结果经检测共获得3.96×106个重组子,重组率>93%,文库经扩增后滴度为3.92×109pfu/ml。结论用SMART方法构建的原发型肝癌组织cDNA文库质量较高,为以后筛选获取原发型肝癌特异抗原基因或其他相关基因奠定了基础。
Objective To construct a hybrid cDNA library of primary hepatocellular carcinoma tissue. Methods Total RNA was isolated from human primary hepatocellular carcinoma (HCC) tissues. The first strand of cDNA was synthesized by oligo (dT) primer containing restriction endonuclease sfiIB and the SMART oligonucleotide containing restriction endonuclease SfiIA was used as primer. Double-stranded cDNA was amplified by PCR. The double-stranded cDNA was digested with sfiI (IA & IB), and cD-NA was fractionated on a CHROMA SPIN-400 column. The cDNA fragments were collected and purified. Then, the cDNA fragments were ligated into λTriplEx2 vector Phage cDNA library. Results A total of 3.96 × 106 recombinants were obtained and the recombination rate was> 93%. The amplified titer of the library was 3.92 × 109 pfu / ml. Conclusion The high quality cDNA library of primary hepatocellular carcinoma constructed by SMART method will provide the foundation for the future screening of primary hepatocellular carcinoma specific antigen genes or other related genes.