携带eGFP及人endostatin-K5的嵌合腺病毒载体Ad5/11的构建及其体外实验研究  

Study on construction of chimeric adenovirus vector Ad5/11 carrying human eGFP and endostatin-K5 and its experimental investigation in vitro

作　　者：王东阳[1] 刘世海[1] 李星[1] 赵俊丽[1] 陈皓[1] 冯飞雪[1] 王丽娴[1] 夏海滨[1] 

机构地区：[1]陕西师范大学生命科学学院基因治疗研究室,陕西西安710062

出　　处：《细胞与分子免疫学杂志》2011年第2期143-145,149,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30872993);陕西省自然科学基金资助项目(2007C201)

摘　　要：目的:构建携带报告基因eGFP及人内皮他丁en-dostatin-K5的嵌合型腺病毒载体Ad5/11。方法:采用重叠PCR及在细菌E.coli.BJ5183中同源重组的方法,获得表达eGFP报告基因的嵌合腺病毒骨架载体,然后以经PacI线性化的嵌合腺病毒载体骨架与PacI线性化的表达endostatin-K5的腺病毒E1穿梭载体共转染真核细胞HEK293细胞中获得携带报告基因eGFP及人endostatin-K5的嵌合型腺病毒载体Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP。以获得的嵌合型腺病毒载体感染U87MG细胞,在荧光显微镜观察eGFP的表达,采用RT-PCR检测endostatin-K5的表达;将所构建的嵌合病毒载体Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP与未经修饰的腺病毒载体Ad5-E1-CMV-eGFP在体外感染人胶质瘤细胞系A172及乳腺癌细胞系MDA-MB-231;通过荧光蛋白eG-FP的表达,比较它们对以上肿瘤细胞的感染效率。结果:嵌合病毒载体Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP可成功地表达eGFP及endostatin-K5。以经过修饰的嵌合型腺病毒载体Ad5/11-E1-CMV-endo-K5/E3-CMV-eGFP感染人脑胶质瘤细胞系A172及人乳腺癌细胞MDA-MB-231的感染效率,明显高于对照未经修饰的腺病毒载体Ad5-E1-CMV-eGFP。结论:携带eGFP及人endostatin-K5的嵌合型腺病毒载体Ad5/11,可明显提高对人脑胶质瘤细胞系A172及人乳腺癌细胞系MDA-MB-231的感染效率。AIM： To construct chimeric adenoviral vector Ad5/ll carrying reporter gene eGFP and human endostatin-K.5. METHODS： Chimeric adenoviral backbone vector expressing eGFP was generated by overlap PCR and homologous recombination in E. coli BJ5183. Then chimeric adenoviral vector AdS/ll-E1-CMV-endo-KS/E3-CMV-eGFP carrying eGFP and human endostatin-K.5 was constructed by co-transfecting Pac I linearized chimeric adenoviral backbone and adenoviral E1 shuttle vector expressing human endostatin-K5 into HEK 293 cells. The expression of eGFP was observed under fluorescent microscope. The expression of human endostain-K.5 in U87MG cells infected by chi- meric adenoviral vector was detected by RT-PCR. The infection efficiency between chimeric adenovirus and unmodified control adenovirus for human glioblastoma cell line A172 and breast cancer cell line MDA-MB-231 in vitro was evaluated by the comparison of the expression of eGFP. RESULTS： Chimeric adenovirus AdS/ll-E1-CMV-endo-KS/E3- CMV-eGFP could successfully express eGFP and endostatin-KS. Chimeric adenoviral vector significantly enhances the infection efficiency for human glioblastoma cell line A172 and breast cancer cell line MDA-MB-231 compared with unmodified adenoviral vector AdS E1-CMV-eGFP. CONCLUSION：Chimeric adenoviral vector Ad5/I]-E]-CMV-endo-KS/E3- CMV-eGFP can significantly improve the infection efficiency for human glioblastoma cell line A172 and breast cancer cell line MDA-MB-231.

关 键 词：endostatin—K5 腺病毒载体 肿瘤 基因治疗 

分 类 号：Q291[生物学—细胞生物学]