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

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

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。

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.