复制缺陷型腺病毒载体介导hVEGF cDNA在C2C12和NIH3T3细胞中的表达  

Recombinant Adenovirus Mediated in vitro hVEGF Gene Tranferring to C2C12 and NIH3T3 Cells

作　　者：孙欲晓[1] 陈诗书[1] 

机构地区：[1]上海第二医科大学生物化学教研室分子生物学实验室人类基因治疗研究中心,上海200025

出　　处：《中国生物化学与分子生物学报》2001年第6期704-709,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金 (No .39870 30 8)资助~~

摘　　要：为提高hVEGFcDNA在心肌中的表达 ,降低在其它组织中表达可能产生的不良反应 ,用PCR法得到人磷酸肌酸激酶 (MCK)增强子和CMV核心启动子 .利用克隆技术构建了含双拷贝MCK增强子 (m)和人巨细胞病毒 (CMV)核心启动子 (c)的嵌合启动子 (mmc) ,构建真核表达质粒pK3 mmcLacZ ,制备受mmc嵌合启动子调控的重组腺病毒Ad mmcVEGF .经X gal染色、β 半乳糖苷酶定量分析、RT PCR、hVEGFELISA定量分析、VEGF活性测定等研究表明 。In order to improve the expression of hVEGF165 cDNA in myocardium and decrease its expression which may result in harmful reaction in other organs,the fragments of human muscle creatine kinase enhancer and CMV core promoter were prepared by PCR with specific primers. Two copies of muscle specific MCK enhancer( m ) and a CMV core promoter( c )were ligated to form the the muscle specific promoter chimera( mmc ), then the plasmid pK3 mmcLacZ was constructed.The recombinant adenovirus (Ad mmc VEGF) was generated by homologous recombination in bacteria and package in 293 cells. By the detection of X gal stain, RT PCR, ELISA, Mile and CAM assays, the results showed that β galactosidase and hVEGF expression were higher in C2C12 myotube than those in NIH3T3 or C2C12 myoblast, which suggested that the chimeric promoter( mmc ) had muscle specific expression function in vitro .

关 键 词：人磷酸肌酸激酶增强子 嵌合启动子 肌细胞 特异性表达 血管内皮生长因子 腺病毒载体 心肌缺血 基因治疗 复制 

分 类 号：Q78[生物学—分子生物学] R394.8[医药卫生—医学遗传学]