论文部分内容阅读
目的:用肝组织特异性启动子,实现人CD81分子在小鼠肝癌细胞Hepa 1-6中的稳定表达。方法:从能感染丙型肝炎病毒(HCV)的人肝癌细胞系HepG2中提取RNA,用随机引物合成cDNA的第1条链,然后用人CD81基因的特异性引物进行PCR扩增,克隆PCR扩增片段。将经测序证明为人CD81基 因的正确序列,连接到肝特异性启动子的下游,使CD81基因的3’端与SV40 polyA加尾序列融合,得到肝组织特异性表达的人CD81融合基因片段。将该融合基因插入真核表达载体pcDNA3中,转染小鼠肝癌细胞系Hepa 1-6。经G418加压筛选后,分别用RT-PCR检测人CD81 mRNA的转录,用FACS检测人CD81蛋白的表达。结果:克隆序列的测序结果与GenBank中登录的序列进行对比表明,得到了人CD81 cDNA的正确序列。构建的人CD81肝特异性融合基因片段可稳定转染Hepa 1-6细胞,并可检测到人CD81在mRNA水平上的转录和在蛋白质水平上的稳定表达。结论:由于CD81是HCV的感染受体,稳定表达HCV感染受体-人CD81分子的小鼠肝癌细胞克隆的获得,为今后研究HCV包膜蛋白与CD81的相互作用、筛选感染阻断药物,以及发展HCV感染的小鼠动物模型奠定了基础。
OBJECTIVE: To achieve stable expression of human CD81 molecule in Hepatoma Hepa 1-6 cells with liver tissue-specific promoter. Methods: RNA was extracted from human hepatocellular carcinoma cell line HepG2 infected with Hepatitis C virus (HCV). The first strand of cDNA was synthesized with random primers and then amplified by PCR using specific primers of human CD81 gene. The PCR amplification Fragment. The correct sequence of human CD81 gene was confirmed by sequencing and ligated downstream of the liver-specific promoter. The 3 ’end of CD81 gene was fused with the SV40 polyA tail sequence to obtain a liver tissue specific human CD81 fusion gene fragment. The fusion gene was inserted into the eukaryotic expression vector pcDNA3 and transfected into mouse hepatoma cell line Hepa 1-6. After pressurized screening with G418, the transcription of human CD81 mRNA was detected by RT-PCR and the expression of human CD81 protein by FACS. Results: Comparison of sequencing results of cloned sequences and sequences registered in GenBank showed that the correct sequence of human CD81 cDNA was obtained. The constructed human CD81 liver-specific fusion gene fragment stably transfected Hepa 1-6 cells, and human CD81 transcription at the mRNA level and stable expression at the protein level were detected. Conclusion: Since CD81 is the HCV infection receptor, we obtained clones of mouse hepatocellular carcinoma cells which stably express human CD81, the receptor of HCV infection, so as to study the interaction between HCV envelope protein and CD81 in the future, The development of animal models of HCV infection in mice laid the foundation.