摘要
目的:应用重叠区扩增基因拼接法(gene SOEing)获得HBV X-HCV C融合基因,构建HBV X-HCV C融合基因重组腺病毒。方法:应用gene SOEing法扩增HBV X-HCV C融合基因并将其克隆至穿梭质粒pAdTrack-CMV,获得重组腺病毒穿梭质粒pAdTrack-CMV-XC,经PmeⅠ酶切线性化后电转化BJ/AdEasy菌,构建重组腺病毒质粒pAd-XC;鉴定正确的pAd-XC经Pac I酶切线性化后脂质体法转染293细胞进行包装,扩增,利用绿色荧光蛋白(Green fluorescent protein,GFP)监测病毒滴度和感染效率,Western-blot法检测目的蛋白的表达。结果:测序结果显示,pAdTrack-CMV-XC构建成功;PCR及Pac I酶切鉴定结果表明pAd-XC构建成功;经Pac I酶切线性化的pAd-XC转染293细胞后24 h即可观察到GFP的表达;回收的病毒可重复感染293细胞,Western-blot法检测到目的蛋白的表达,证实有感染能力且可表达目的蛋白的病毒颗粒包装成功。结论:HBV X-HCV C融合基因重组腺病毒构建成功,为进一步研究HBV X蛋白和HCV C蛋白的生物学功能奠定了基础。
Objective： To construct the recombinant adenovirus that express HBV X- HCV C fusion gene for further study. Methods： The HBV X - HCV C fusion gene was amplified by using gene SOEing and was inserted into the multiple clone site of pAdTraek- CMV. The linearized shuttle plasmid was homogenously recombined with AdEasy- 1 in BJ5183. The candidate clone was analyzed by restriction endonuelease digestion, PCR, and sequence scan, then the reeombined adenovirus plasmid was digested with Pae Ⅰ and transfeeted into 293 cells for packaging and amplifying. Infection titer and rate were monitored by green fluorescent protein （GFP） expression. The expression of HBV X- HCV C fusion protein was detected by Western- blot. Results ： With restriction endonuelease analysis and PCR methods , it has been confirmed that HBV X - HCV C fusion gene was cloned into the adenovirus vector successfully. The expression of fusion protein was detected by Western- blot. Conclusion： The reeombined adenovirus containing HBV X - HCV C fusion gene was constructed successfully, which would contribute to the advanced functional study of HBV X and HCV C fusion protein.
出处
《内蒙古医学杂志》
2007年第9期1043-1047,共5页
Inner Mongolia Medical Journal
