携带丙型肝炎病毒非结构蛋白3基因的重组腺病毒构建研究

目的构建表达丙型肝炎病毒(HCV)非结构蛋白3 (NS3)的复制缺陷型重组腺病毒,为防治HCV感染的基因免疫和基因治疗提供实验基础. 方法将HCV NS3基因定向克隆到穿梭质粒pAdTrack-CMV上,经与腺病毒骨架质粒pAdEasy-1在大肠埃希菌BJ5183内同源重组后,转染293细胞进行包装,并反复感染293细胞进行扩增. 结果通过PCR扩增、酶切、核酸测序及Western杂交法检测鉴定,均证实插入穿梭质粒中的片段为HCV NS3基因(基因型1b);所包装出的复制缺陷型重组腺病毒AdEasy-GFP-NS3具有良好的感染能力,并可在293细胞中表达HCV NS3蛋白. 结论 AdEasy-GFP-NS3携带有HCV NS3基因,能有效地感染293细胞并高效表达,从而为丙型肝炎基因疫苗的进一步研究奠定了基础.

Construction of Recombinant Adenovirus Carrying Hepatitis C Virus Nonstructural Protein 3 Gene

OBJECTIVE To construct the replicate-deficient recombinant adenovirus expressing nonstructural(protein) 3(NS3) of hepatitis C virus(HCV) for gene therapy against HCV infection.METHODS pAdTrack-NS3 was cloned by inserting the fragment of HCV NS3 into the shuttle plasmid vector pAdTrack-CMV.For getting(recombinant) adenovirus plasmid(pAdEasy-GFP-NS3),the homologous recombination with pAdTrack-NS3 and the backbone plasmid pAdEasy-1 took place in bacteria BJ5183;then pAdEasy-GFP-NS3 transfected in 293 cells for getting packaged infectious replicate-deficient recombinant adenovirus and were amplified by infecting 293 cells.RESULTS By PCR amplification,restricted enzymatic digestion,DNA sequencing and Western blot,the fragment inserted within pAdTrack-NS3 was certificated as HCV NS3(genotype 1b).AdEasy-GFP-NS3 could(efficiently) infect 293 cells and express NS3 protein.CONCLUSIONS Replicate-deficient recombinant adenovirus AdEasy-GFP-NS3 could infect 293 cells and(express) HCV NS3 protein(effectively,) which would provide(experimental) basis for gene immunization against HCV infection.