【摘要】 目的构建一种同时携带人肝细胞生长因子(hHGF)及绿色荧光蛋白(GFP)的腺病毒重组体,为hHGF的基因治疗提供实验基础。方法对重组质粒pcDNA3-hHGF进行酶切鉴定及测序正确后,酶切获得hHGF,亚克隆至带有GFP标记的穿梭质粒pAdTrack-CMV上。选取hHGF正向插入的重组穿梭质粒经PmeⅠ充分线性化后,与骨架质粒pAdEasy-1在大肠杆菌BJ5183中同源重组,挑取阳性克隆行PCR及酶切鉴定。重组病毒质粒用PacⅠ酶切线性化后,脂质体转染HEK293细胞进行包装并扩增获取重组病毒上清,RT-PCR及WesternBlot方法检测hHGF的表达。结果目的基因的测序结果与Genebank上的hHGF序列一致。hHGF基因插入重组腺病毒载体的预期位置,感染Ad-hHGF的HEK293细胞中可检测到hHGFmRNA和蛋白的表达。结论成功构建了同时携带hHGF和GFP的重组腺病毒表达载体Ad-hHGF,为进一步探讨hHGF的生物学功能提供了实验依据。更多还原

【Abstract】 Objective To construct recombinant adenovirus vector encoding both human hepatocyte growth factor (hHGF) and green fluorescent protein (GFP) in order to provide the experimental basis for hHGF gene therapy.Methods The target gene of hHGF was cloned from plasmid pcDNA3-hHGF and was verified by DNA sequence analysis.The hHGF gene was subcloned to shuttle vector pAdTrack-CMV which had GFP in it.The right insert of hHGF in the shuttle vector was identified by restriction enzymes.Plasmid pAdTrack-hHGF was linearized by digestion with restriction endonuclease PmeⅠ and subsequently cotransformed into E.coli BJ5183 cells with adenoviral backbone plasmid pAdEasy-1 to make homologous recombination.After linearized by PacⅠ,the recombinant virus plasmid was transfected into HEK293 cells by Lipofectamin2000 and was packaged and amplified in HEK293 cells.The expression of hHGF was detected by RT-PCR and Western Blot.Results The hHGF sequence in plasmid pcDNA3-hHGF was the same as the one which provided by Genebank.The hHGF gene was inserted into the expect position of recombinant adenovirus vector.After infection,the expression of hHGF mRNA and protein could be detected in HEK293 cells.Conclusions The recombinant adenovirus vector Ad-hHGF encoding both hHGF and GFP has been constructed successfully,which provides experimental basis for further studying the biological function of hHGF.更多还原