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目的:构建能表达HCVC基因的复制缺陷型腺病毒表达载体.方法:将HCVH株C区基因定向插入到腺病毒穿梭质粒pAd.CMV-Link.1中,获得重组质粒pAd.HCV-C,再与pJM17共转染293细胞,包装腺病毒表达载体.通过酶切、PCR及测序对穿梭质粒进行了鉴定.对腺病毒载体进行了感染性鉴定、电镜鉴定及双引物PCR鉴定.利用间接免疫荧光法和Westernblot检测了腺病毒载体在人肝癌细胞HepG2中的表达.结果:酶切、PCR及测序鉴定证实,穿梭质粒插入片段为HCVC区基因.包装的腺病毒载体具有良好的感染性,可以在293细胞中形成病毒颗粒,腺病毒载体内携带HCVC区基因,并可以在HepG2细胞中表达HCVC抗原.结论:包装成功的复制缺陷型腺病毒载体可以在HepG2细胞中表达HCVC抗原,为丙型肝炎的基因治疗及疫苗的进一步研究奠定了基础.
Objective: To construct replication-defective adenovirus expression vector expressing HCVC gene.Methods: The C region of HCVH strain was inserted into the adenovirus shuttle plasmid pAd.CMV-Link.1 to obtain the recombinant plasmid pAd.HCV-C 293 cells were cotransfected with pJM17 and packaged into adenovirus expression vector.The shuttle plasmid was identified by restriction enzyme digestion, PCR and sequencing.Adenovirus vector was identified by infection, electron microscopy and double-primer PCR.Immunofluorescence Western blot was used to detect the expression of adenovirus vector in HepG2 cells.Results: Enzyme digestion, PCR and sequencing proved that the shuttle plasmid insert gene was HCV C region.The packaged adenovirus vector had good infectivity, 293 cells.The recombinant adenoviral vector can express HCVC gene in HepG2 cells and express HCVC antigen in HepG2 cells.Conclusion: The packaging-defective adenovirus vector can express HCVC antigen in HepG2 cells, The gene therapy and vaccine further laid the foundation for further research.