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目的构建携带肝细胞生长因子HGF的重组腺病毒表达载体。为研究HGF的功能、作用机制及临床应用奠定基础。方法以pcDNA3.0-HGF为模板,经PCR扩增得到HGF基因,将其插入到带有绿色荧光蛋白基因的穿梭载体pAdTrack-CMV-GFP中,得到重组质粒pAdTrack-CMV-HGF-GFP。将线性化的pAdTrack-CMV-HGF-GFP与pAdEasy-1质粒共转化BJ5183感受态细胞,进行同源重组,得到重组腺病毒质粒,经PacI线性化后,转染AD293细胞。结果得到重组腺病毒,转染重组腺病毒DNA的AD293细胞经荧光显微镜能观察到AD293细胞内有绿色荧光,且出现细胞病变反应(cytopathic effects,CPE),对传代的Ad-HGF进行PCR分析证实得到目的基因,感染CHL细胞,通过western blotting分析HGF蛋白表达量,从而证实重组腺病毒载体构建成功。结论腺病毒制备简便,成本低,周期短,毒性小,产量高,感染率及蛋白表达率高,成功构建Ad-HGF为进一步研究HGF并最终使其在临床应用中更为方便提供实验基础。
Objective To construct a recombinant adenovirus vector carrying hepatocyte growth factor HGF. To lay the foundation for studying the function, mechanism and clinical application of HGF. Methods HGF gene was amplified by PCR using pcDNA3.0-HGF as a template and inserted into the shuttle vector pAdTrack-CMV-GFP with green fluorescent protein gene to obtain the recombinant plasmid pAdTrack-CMV-HGF-GFP. The linearized pAdTrack-CMV-HGF-GFP and pAdEasy-1 plasmid were co-transformed into BJ5183 competent cells for homologous recombination to obtain the recombinant adenovirus plasmid, which was linearized with PacI and transfected into AD293 cells. RESULTS: AD293 cells transfected with recombinant adenovirus DNA showed green fluorescence in AD293 cells with cytopathic effects (CPE) by fluorescence microscopy and PCR analysis of passaging Ad-HGF confirmed The target gene was obtained and infected with CHL cells. The expression of HGF protein was analyzed by western blotting, which confirmed the successful construction of the recombinant adenovirus vector. Conclusions Ad-HGF was successfully constructed to facilitate the study of HGF and to provide an experimental basis for its convenience in clinical application. The adenovirus has the advantages of simple preparation, low cost, short cycle, low toxicity, high yield, high infection rate and high protein expression rate.