TCF25干扰慢病毒载体构建与鉴定.docVIP

TCF25干扰慢病毒载体构建与鉴定 　　摘 要 为构建一个针对TCF25（transcription factor 25）基因的慢病毒干扰载体， 设计并合成3组针对TCF25的短发夹RNA序列 （shRNA），通过基因重组技术连入 pLL3.7载体，酶切鉴定及DNA测序后，重组正确的pLL3.7质粒与病毒包装质粒共转染293FT细胞，培养48 h后，分别收集细胞培养上清液，感染H9C2细胞，Westernblot检测TCF25在H9C2细胞中的表达.结果显示TCF25蛋白在H9C2细胞中的表达被抑制.这说明TCF25的慢病毒干扰载体构建成功. 　　关键词 TCF25基因；293FT细胞；H9C2 细胞；慢病毒载体 　　中图分类号 Q786文献标识码 A文章编号2016 　　Construction and Characterization of a Lentiviral Vector for RNA Interference of TCF25 　　LI Youfeng， PENG Hao， GAO Jianfang， GAO Jing， CHEN Yu， WU Xiushan， LI Yongqing* 　　（Center for Heart Department， Key lab of MOE for Department Biology and Protein Chemistry， 　　Hunan Normal University， Changsha 410005， China） 　　Abstract Objective To construct a lentiviral vector for RNA interference of TCF25 （transcription factor 25） gene and identify it. Methods Three pairs of complementary short hairpin RNA （shRNA） oligonucleotides targeting the TCF25 gene were designed， synthesized， and then inserted into pLL3.7 vectors by gene recombination technology. The recombinant plasmid was identified by enzyme digestion and DNA sequencing. Correct recombinant plasmids were cotransfected with packaging plasmids into 293FT cells. The cell culture supernatant was obtained after 48 hours， and then applied to infect H9C2 cells. The expression of TCF25 in H9C2 cells was detected by westernblot. Results The recombinant plasmid was successfully constructed. The westernblot showed that the expression of TCF25 in H9C2 cells was inhibited after the cells were successfully infected with the lentiviral vector supernatant. Conclusion The lentiviral vector interfering TCF25 was constructed successfully. 　　Key words TCF25 gene； 293FT cell； H9C2 cell； lentiviral vector 　　TCF25（transcription factor 25）是在小鼠胚胎发育过程中广泛表达的一个含有bHLH结构域的转录因子，且其C端还含有一个功能未知的DUF654结构域.根据TCF25广泛的表达模式和包含的转录激活结构域，推测其可能在脑和心脏等各种器官发育过程中发挥重要的调控作用[13].同时，已有研究表明TCF25作为SRF信号通路的抑制因子调控基因的表达，且能够抑制P19CL6细胞向心肌细胞分化[4]，但其具体作用机制需要继续探索. 　　RNA干扰（RNA interference，缩写RNAi）是指在进化过程中高度保守的双链RN