hTERT启动子调控的SEA-CD80基因共表达重组腺病毒载体的构建及在不同肿瘤细胞中的表达鉴定

Construction of recombinant adenovirus of SEA and CD80 genes co- expression regulated by human TERT promoter and its expression in different tumor cells

目的:构建肿瘤靶向性葡萄球菌肠毒素A和CD80基因共表达重组腺病毒载体。方法:采用PCR技术从质粒pShuttle-AFP-CD80扩增人CD80全长cDNA片段,克隆至已构建载体pMD18-T-BIS,取代小鼠CD80 cDNA片段。重组质粒命名为pMD18-T-hBIS。经酶切,将CWV增强子修饰的人端粒酶反转录酶启动子从已构建载体pGL3-CWVE-hTP,亚克隆至穿梭质粒pShuttle2,然后经酶切将片段hCD80-IRES-SEA从pMD18-T-hBIS质粒亚克隆至CWV增强子修饰的人端粒酶反转录酶启动子下游,构建质粒pShuttle2-CE-hTP-hBIS。再与腺病毒骨架质粒pAdEasy-1共转化E.coli BJ5183,以获得的重组子(命名为Ad-CE-hTP-BIS)转染Ad293细胞制备病毒并纯化。将病毒分别感染人肝癌细胞H7721、宫颈癌细胞株Hela细胞和原代培养的人牙龈成纤维细胞,经免疫荧光染色后,激光共聚焦显微镜观察SEA和CD80在细胞膜的表达情况。结果:重组腺病毒能够使CD80和SEA在人肝癌细胞H7721、宫颈癌细胞株Hela细胞膜上共表达,而在人牙龈成纤维细胞中不表达。结论:成功地构建了多肿瘤靶向性SEA和CD80基因共表达重组腺病毒载体。为应用SEA和CD80基因对人多种肿瘤进行靶向基因治疗奠定了基础。

To construct tumor - targeting recombinant co - expression adenovirus vector of Staphylo-coccal enterotoxin A（SEA）and CD80 gene. Methods：Complete human CD80 cDNA was amplified by PCR from pShuttle - AFP - CD80 plasmid and cloned into pMD18 - T - BIS plasmid to replace mouse CD80 cDNA. The recom-binant plasmid was named as pMD18 - T - hBIS. CMV enhancer - modified human telomerase reverse transcriptase （hTERT）promoter was subcloned into pShuttle2 plasmid from pGL3 - CWVE - hTP,then hCD80 - IRES - SEA frag-ment was subcloned into it downstream of CMV enhancer - modified hTERT promoter from pMD18 - T - hBIS by re-striction enzyme digestion to construct pShuttle2 - CE - hTP - hBIS plasmid. The pShuttle2 - CE - hTP - hBIS plas-mid was cotransformed into E. coli BJ5183 with backbone vector pAdEasy - 1 to obtain recombinant adenovirus DNA named Ad - CE - hTP - BIS. The recombinant adenovirus DNA was transfected into 293 cells to prepare adenovirus. After human hapatoma cell line H7721,cervical cancer cell lines Hela and human gingival fibroblasts（HGF）were in-fected by recombinant adenovirus,the expression of SEA and CD80 on the surface of cells was observed by immuno-fluorescent staining and laser confocal microscope. Results：SEA and CD80 was specifically co - expressed on the sur-face of infected H7721 and Hela cells but not on HGF cells. Conclusion：Different tumors - targeting recombinant co- expression adenovirus vector of SEA and CD80 gene was successfully constructed,which lays the foundation for fur-ther research on application of SEA and CD80 in targeted gene therapy for different tumors.