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Construction,identification and expression of three kinds of shuttle plasmids of adenovirus expression vector of hepatitis C virus structure gene
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- Author:
- No author available
- Journal Title:
- CHINESE CRITICAL CARE MEDICINE
- Issue:
- 2
- DOI:
- 10.3760/j.issn:1003-0603.2003.02.002
- Key Word:
- 丙型肝炎病毒;结构基因;腺病毒;表达载体;穿梭质粒
Abstract： 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 Ⅱ及 Hind Ⅲ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pBRTM/HCV1-3011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 Ⅱ及 Hind Ⅲ双酶切,定向插入到腺病毒穿梭质粒pAd.CMVLink.1中CMV启动子下游Bgl Ⅱ与 Hind Ⅲ位点之间,获得3种重组腺病毒穿梭质粒pAd.HCV-C、 pAd.HCVCE1和 pAd.HCV-S(C+E1+E2).通过Bgl Ⅱ/Hind Ⅲ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HepG2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HepG2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.
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