Txt5腺病毒载体构建及其在心肌细胞中的表达  

Construction of Txt5 Adenovirus Vector and Analysis of Its Expression in Cadiomyocytes

作　　者：彭浩[1] 陈宇[1] 李佑锋[1] 朱旋[1] 周作琼 吴秀山[1] 范雄伟[1] 李永青[1] 

机构地区：[1]湖南师范大学心脏发育研究中心蛋白质化学及鱼类发育生物学教育部重点实验室,湖南长沙410081

出　　处：《怀化学院学报》2015年第5期45-48,共4页Journal of Huaihua University

基　　金：国家自然科学基金资助项目(81270156;81400304;81470377);湖南省社会发展支撑计划项目(2014SK3009;2015JJ3087);湖南省生物发育工程及新产品研发协同创新中心(2013-448-6)

摘　　要：设计小鼠Txt5基因的特异性引物,将小鼠c DNA作为模板,用PCR扩增出m Txt5编码区,并加入HA标签序列.将这一片段插入p MD-18T载体,再亚克隆至p Adtrack-cmv穿梭载体上.线性化后,转化BJ5183感受态细胞,在BJ5183中发生同源重组获得p Ad-Txt5质粒.p Ad-Txt5线性化后转染293A细胞,包装得到含Txt5基因的病毒.将病毒溶液感染大鼠原代心肌细胞,一段时间后观察绿色荧光,然后通过RT-PCR和免疫印迹法检测HA标签蛋白的表达.结果表明成功构建小鼠Txt5腺病毒表达载体并实现在大鼠原代心肌细胞中的表达.Specific primers for the mouse Txt5 gene were designed and employed to amplify the coding sequence using mouse cDNAs as the template .HA - tagged sequence was induced into the PCR primers .The PCR product was inserted into the pMD -18T vector and subcloned into pAd - track - cmv shuttle vector .The recombinant plasmid was linearized and transformed into bacteria BJ5183 .After homologous recombination in the bacteria ,plasmid pAd - Txt5 was obtained .Linearized pAd - Txt5 was transfected into 293 A cells .Then , the recombinant virus containing Txt5 was obtained after packaging . Cardiomyocytes were infected with the collected virus fluid .To detect the expression of Txt5 ,green fluorescence protein （GFP） was observed by green fluorescence and RT - PCR , immunoblotting was performed . The results showed that mouse Txt5 adenovirus vector was constructed successfully ,and it could induce the sequence coding Txt5 - HA into cardiomyocytes .

关 键 词：腺病毒 Txt5 心肌细胞 

分 类 号：Q786[生物学—分子生物学]