丙型肝炎病毒基因免疫重组真核表达质粒构建的初步研究

目的:利用真核表达载体pCDNA3.1(+)与HCV核心区基因重组,为进一步表达蛋白打基础。方法:首先从1例HCV感染者血清中用反转录-PCR法获得全长的核心区基因并测序,继而将其克隆到原核表达载体pQE-30上并使之在大肠杆菌中得到表达,然后将核心区基因与真核表达载体pCDNA3.1(+)重组,构建重组体。结果:HCV核心区基因为HCV型,将其克隆到原核表达载体pQE-30上并使之在大肠杆菌中得到表达,分子量为22KD,表达量占菌体蛋白的8.7%。然后将核心区基因与真核表达载体pCDNA3.1(+)重组,构建了pCDNAHCVc191和pCD-NAHCVc-hIL2两种重组体。结论:pCDNAHCVc191和pCDNAHCVc-hIL2两种重组体,尤其后者是核心区基因与人白介素-2基因融合,可引发更好的基因免疫效果。上述工作奠定了整个HCV基因免疫研究的基础。

Primary study of construction of eukaryotic expression plasmid for genetic immunization against HCV

Objective: To produce the recombination of the hepatitis C virus in eukaryotic expression plasmid.Methods:We isolate the whole HCV core gene from a serum sample of a Chinese infected by HCV via RT-PCR,sequence and express it in E.coli,then subclone it into pDNA3.1( ) mammalian expression vector and construct recombinant plasmid.Results:The whole HCV core gene was cloned into prokaryotic expression vector pQE-30 and expressed in E.coli,the quantitative of expression protein were 8.7% of whole thallus protein.Then it was subcloned into pDNA3.1( ) and construct two recombinant plasmid which can express foreign proteins in cultured cells and animals with different forms,one called pCDNAHCVc191,another pCDNAHCVc-hIL2.Conclusion:They are bases for further investigation as potential genetic vaccines against HCV infection.