摘要
目的构建能表达HVC基因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HCVC基因的腺病毒载体做准备。方法用分别含有BgⅡ及HindⅢ酶切位点的HCVC区基因上、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HCVC区基因片段,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与HindⅢ位点之间,获得重组表达质粒pAd.HCV-C通过BglⅡ/HindⅢ双酶切、PCR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-C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-C插入片段为HCVC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-C可以在7721细胞中瞬时表达HCVC区基因,为包装表达HCVC基因的腺病毒载体奠定了基础。
AIM To construct a recombinant framework plasmid of adenovirus expression vector which can express HCV core gene. This should be useful to pack adenovirus expression vector which can express HCV core gene effectively. METHODS The HCV core gene derived from the plasmid pBRTM/HCV 1-3011 by using polymerase chain reaction (PCR) was inserted into the backward position of cytomegalovirus (CMV) immediate early promotor element of framework plasmid (pAd. CMV-Link. 1) of adenovirus expression vector, then the recombinant plasmid pAd. HCV-C was obtained. The pad. HCV-C was identified by endonuclease, PCR and sequencing. HCV core gene was expressed transiently with Lipofectamine coated in human hepatoblastoma 7721 cells which examined by indirect immunofluorescence. RESULTS Insert DNA of pAd. HCV-C was confirmed HCV core gene by endonuclease, PCR and sequencing, pAd. HCV-C can express HCV core gene transiently in 7721 cells which confirmed by indirect immunofluorescence. CONCLUSION Recombinant framework plasmid of adenovirus expression vector pAd. HCV-C can express HCV core gene transiently in 7721 cells. This should be useful to pack adenovirus expression vector which can express HCV core gene.
出处
《世界华人消化杂志》
CAS
2001年第6期635-639,共5页
World Chinese Journal of Digestology
基金
国家自然科学基金
No.39800122
关键词
C型肝炎样病毒属
遗传学
病毒基因
腺病毒科
遗传载体
hepatitis C-like viruses/genetics
genes, viral
adenoviridae/genetics
genetic vectors
plasmids
recombination genetic
