【摘要】 目的构建HCV C基因重组腺病毒,以期用于HCV C蛋白的功能研究。方法以含有丙肝病毒1b亚型核心基因的质粒pcDNA3.1/HCV-C为模板,PCR扩增HCV C基因,PCR产物双酶切后亚克隆至穿梭质粒pAdTrack-CMV上,与骨架质粒pAdEasy-1在BJ5183细菌内同源重组,获得重组腺病毒质粒pAd-C,PacⅠ酶切线性化后脂质体法转染293细胞进行包装,获得重组腺病毒Ad-C,继之在293细胞内扩增,利用报告基因GFP监测病毒滴度和感染效率,Western blot检测HCV C蛋白的表达。结果PCR及Western blot结果均证实重组腺病毒Ad-C构建成功。结论成功构建了重组腺病毒Ad-C,为进一步研究核心蛋白的生物学功能奠定了基础。更多还原

【Abstract】 Objective To construct the recombinant adenovirus containing HCV C gene for functional study on HCV C protein.Met-(hods The) HCV C gene was amplified by PCR from the pcDNA3.1/HCV-C,then the fragment was inserted into the multiple clone site of pAdTrack-CMV.The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183.The candidate clone was analyzed by restriction endonuclease digestion,PCR,and sequence scan.The recombined adenovirus plasmid was digested with PacⅠ and transfected into 293 cells for packaging and amplifying.Infection titer and rate were evaluated through monitoring green fluorescent protein(GFP) expression.The expression of C protein was detected by Western blotting.Results Restriction endonuclease analysis and PCR demonstrated that HCV C gene was cloned into the adenovirus vector successfully.Conclusion The recombined adenovirus Ad-C is constructed successfully,which would contribute to the advanced functional study of HCV C protein.更多还原