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用分别含有BglⅡ及HindⅢ酶切位点的HCVCE1基因上、下游引物 ,以含有HCVH株基因序列的质粒 pBRTM/HCV1 30 11为模板 ,通过PCR扩增获得HCVCE1基因片段 ,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 CE1。通过BglⅡ /HindⅢ双酶切、PCR及插入片段序列测定对质粒进行鉴定 ,证实了 pAd .HCV CE1插入片段为HCVCE1区基因片段。以抗HCVC单克隆抗体为一抗 ,利用间接免疫荧光法证实 pAd .HCV CE1可以在人肝癌细胞 772 1中瞬时表达。以上结果初步表明 ,构建的质粒 pAd .HCV CE1可以表达HCVCE1区基因 ,为进一步包装能高效表达HCVCE1基因的腺病毒载体奠定了基础。
The HCVCE1 gene fragment was amplified by PCR using the upstream and downstream primers of HCVCE1 gene containing BglII and HindIII restriction sites respectively and the plasmid pBRTM / HCV1 3011 containing the HCVH strain gene sequence as a template, and inserted into the adenovirus backbone plasmid The recombinant plasmid pAd. HCV CE1 was obtained between the BglII and HindIII sites downstream of the CMV promoter in pAd. CMV Link 1. The plasmid was identified by BglⅡ / HindⅢ double digestion, PCR and insert fragment sequencing, which confirmed that the pAd. HCV CE1 insert was HCVCE1 gene fragment. The anti-HCVC monoclonal antibody was used as the primary antibody, and indirect immunofluorescence was used to confirm the expression of pAd.HCV CE1 was transiently expressed in human hepatocellular carcinoma cell line 772 1. The above results preliminarily showed that the constructed plasmid pAd.HCV CE1 can express the HCVCE1 region gene, which lays the foundation for further packaging the adenovirus vector capable of efficiently expressing the HCVCE1 gene.