HCV结构基因腺病毒表达载体骨架质粒pAd.HCV-S的构建、鉴定及表达

目的：构建能表达HCV结构基因（C＋E1＋E2）的腺病毒表达载体的重组骨架质粒，为进一步包装能高效表达HCV结构基因的腺病毒载体做准备，方法：用分别含有BglⅡ及HindⅢ酶切位点的HCV结构基因上、下游引物，以含有HCVH株基因序列的质粒pBRTM/BCV1-3011为模板，通过PCR扩增获得HCV结构基因片段，基因片段回收后，以BglⅡ及HindⅢ双酶切，定向插入到腺病毒骨架质粒pAd.CMV-Link.1中CMV启动子下游BglⅡ及HindⅢ位点之间，获得重组表达质粒pAd.HCV-S.通过BglⅡ／HindⅢ双酶切、PCV及插入片段序列测定对质粒进行了鉴定。以抗HCVC单克隆抗体为一抗，利用间接免疫荧光法检测了pAd.HCV-S在人肝癌细胞7721中的瞬时表达。结果：酶切、PCR及测序鉴定证实，pAd.HCV-S插入片段为HCVCE1＋E2区基因片段，免疫荧光法检测表明其可以在7721细胞中瞬时表达。结论：构建的质粒pAd.HCV-S 可以表达HCV结构基因，为包装表达HCV结构基因的腺病毒载体奠定了基础。

Construction,identification and expression of framework plasmid pAd.HCV-S of adenovirus expression vector of HCV structure gene

Objectives:To construct a recombinant framework plasmid o f adenovirus expression vector which can express HCV structure gene(C+E1+E2). Methods:The HCV structure gene derived from the plasmid pBRTM/HCV1-30 11 by using polymerase chain reaction(PCR) was inserted into the backward positi on of cytomegalovirus(CMV) immediate early promotor element of framework plasmid ( pAd.CMV-Link.1) of adenovirus expression vector,then the recombinant plasmid pA d .HCV-S was obtained.The pAd.HCV-S was identified by endonuclease,PCR and seque nc ing.HCV structure gene was expressed transiently with Lipofectamine 2000 coated in human hepatoblastoma 7721 cells which conformed by immunofluorescence. Results:Insert DNA of pAd.HCV-S was conformed HCV structure gene by endo n uclease,PCR and sequencing.pAd.HCV-S can express HCV structure gene transientl y in 7721 cells which conformed by immunofluorescence. Conclusions:R ecombinant framework plasmid of adenovirus expression vector can express HCV str ucture gene.This should be useful to pack adenovirus expression vector which can express HCV structure gene.