CAJ | 학술논문

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。
목적：구건종류파향성포도구균장독소 A 화 CD80기인공표체중조선병독재체。방법：채용 PCR 기술종질립 pShuttle - AFP - CD80확증인 CD80전장 cDNA 편단，극륭지이구건재체 pMD18- T - BIS，취대소서CD80 cDNA 편단。중조질립명명위 pMD18- T - hBIS。경매절，장 CWV 증강자수식적인단립매반전록매계동자종이구건재체 pGL3- CWVE - hTP，아극륭지천사질립 pShuttle2，연후경매절장편단 hCD80- IRES - SEA종 pMD18- T - hBIS 질립아극륭지 CWV 증강자수식적인단립매반전록매계동자하유，구건질립 pShuttle2-CE - hTP - hBIS。재여선병독골가질립 pAdEasy -1공전화 E. coli BJ5183，이획득적중조자（명명위 Ad - CE- hTP - BIS）전염 Ad293세포제비병독병순화。장병독분별감염인간암세포 H7721、궁경암세포주 Hela 세포화원대배양적인아간성섬유세포，경면역형광염색후，격광공취초현미경관찰 SEA 화 CD80재세포막적표체정황。결과：중조선병독능구사 CD80화 SEA 재인간암세포 H7721、궁경암세포주 Hela 세포막상공표체，이재인아간성섬유세포중불표체。결론：성공지구건료다종류파향성 SEA 화 CD80기인공표체중조선병독재체。위응용 SEA 화 CD80기인대인다충종류진행파향기인치료전정료기출。
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.