携带人肝细胞生长因子腺病毒重组体的构建  

Construction of recombinant adenovirus vector encoding human HGF

作　　者：朱小玉 朱薇波 张祥忠[2] 周育森[3] 陈嘉瑜[4] 孙自敏 陈运贤[2] 

机构地区：[1]安徽医科大学附属省立医院血液内科 [2]中山大学附属第一医院血液内科 [3]军事医学科学院微生物流行病研究所病原分子生物学研究室 [4]广州市第十二人民医院内科

出　　处：《中华临床医师杂志（电子版）》2011年第7期1906-1911,共6页Chinese Journal of Clinicians(Electronic Edition)

基　　金：广州市医药卫生科技项目(201102A212004)

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。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.

关 键 词：肝细胞生长因子 绿色荧光蛋白质类 腺病毒 人 基因疗法 

分 类 号：R450[医药卫生—治疗学]