3种丙型肝炎病毒结构基因腺病毒载体穿梭质粒的构建鉴定及表达

Construction,identification and expression of three kinds of shuttle plasmids of adenovirus expression vector of hepatitis C virus structure gene

目的 :构建能表达 HCV不同结构基因 (C、C+E1、C+E1+E2 )的腺病毒表达载体的穿梭质粒 ,为进一步包装能高效表达 HCV不同结构蛋白的腺病毒载体做准备。方法 :用分别含有 Bgl 及 Hind 酶切位点的 HCV不同片段结构基因上、下游引物 ,以含有 HCV H株基因序列的质粒 p BRTM/HCV130 11为模板 ,通过 PCR扩增获得 HCV 3种不同结构基因片段 ,基因片段回收后 ,分别以 Bgl 及 Hind 双酶切 ,定向插入到腺病毒穿梭质粒 p Ad.CMV L ink.1中 CMV启动子下游 Bgl 与 Hind 位点之间 ,获得 3种重组腺病毒穿梭质粒 p Ad.HCV C、 p Ad.HCV CE1和 p Ad.HCV S(C+E1+E2 )。通过 Bgl /Hind 双酶切、聚合酶链反应 (PCR)及插入片段序列测定对质粒进行了三重鉴定 ,以抗 HCV C单克隆抗体为一抗 ,利用间接免疫荧光法及 Western Blot检测了 3种穿梭质粒在 Hep G2细胞中的瞬时表达。结果 :酶切、PCR及测序鉴定证实 ,3种穿梭质粒的插入片段分别为 HCV C、C+E1和 C+E1+E2区基因片段 ,免疫荧光及 Western Blot法检测表明其可以在 Hep G2细胞中瞬时表达。结论 :构建的 3种腺病毒穿梭质粒分别可以表达 HCV不同段的结构基因 ,为包装表达 HCV不同结构基因的腺病毒载体奠定了基础。

Objective:To construct three recombinant shuttle plasmids of adenovirus expression vector which can express hepatitis C virus(HCV) different structure genes(C,C+E1,C+E1+E2) in order to pack adenovirus expression vectors which can express HCV different structure gene effectively.Methods:The different HCV structure genes derived from the plasmid pBRTM/HCV13011 by using polymerase chain reaction (PCR) were inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of shuttle plasmid(pAd.CMVLink.1) of adenovirus expression vector respectively,then the three recombinant plasmids (pAd.HCVC,pAd.HCVCE1,pAd.HCVS) were obtained.The recombinant plasmids were identified by endonuclease,PCR and sequencing.HCV structure genes were expressed transiently with Lipofectamine 2000 coated in HepG2 cells which were confirmed by immunofluorescence and WesternBlot. Results:Insert DNAs of the three recombinant plasmids' were confirmed to be HCV different structure genes by endonuclease,PCR and sequencing.The three recombinant plasmids can express HCV structure gene (C,C+E1,C+E1+E2) transiently in HepG2 cells which were confirmed by immunofluorescence and WesternBlot. Conclusions:The three recombinant shuttle plasmids of adenovirus expression vector can express HCV structure gene(C,C+E1,C+E1+E2) transiently.This should be useful to pack adenovirus expression vector which can express HCV structure genes.