复制缺陷型HCV C基因腺病毒表达载体的构建包装及鉴定

Construction, package and identification of replication-deficient recombinant adenovirus expression vector of HCV C

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.

AIM:To construct a replication-deficient recombinant adenovirus expression vector of HCV C. METHODS:The HCV core gene was cloned at the down- stream of CMV promoter of the adenoviral shuttle plasmid pAd.CMV-link.1, and the resultant recombinant plasmid pAd. HCV-C was cotransfected into 293 cell together with plasmid pJM17 containing adenoviral genome, then the adenovirus expression vector was obtained, and identified by infecting test,electronic microscope observation and PCR co-amplification. The plasmid pAd.HCV-C was identified by endonuclease, PCR and sequencing.The expressive activity of adenovirus vector was identified by immunofluorescence and Western blot. RESULTS:HCV core gene in the inserted DNA of pAd.HCV-C was confirmed by endonuclease, PCR and sequencing. Results of infecting test, electronic microscopic observation and PCR co-amplification showed that the adenovirus vector had been constructed successfully. Expression of HCV core antigen was proved in the HepG2 cells by immunofluorescence and Western blot.study established a foundation for further study on HCV vaccines and gene therapy for hepatitis C.