MBP序列特异性shRNA质粒载体的构建及鉴定

Construction and identification of MBP sequence-specific shRNA plasmid vector

目的:以pGenesil-1质粒为介导,构建针对MBP基因的shRNA表达载体。方法:设计特异性表达MBP shRNA结构的互补DNA序列3个及1个阴性对照序列,退火并克隆至质粒载体pGenesil-1中,转化DH5α菌株,进行重组质粒的酶切鉴定及测序分析。结果:经酶切鉴定及测序分析,筛选出的重组体测序结果和靶序列相同,成功构建了shRNA重组质粒载体pGenesil-1-MBP-1、pGenesil-1-MBP-2、pGenesil-1-MBP-3及pGenesil-1-MBP-neg。结论:成功构建MBP shRNA表达载体,为进一步研究MBP基因在细胞中的作用机制奠定基础。

Objective：To construct the recombinant plasmids expressing MBP shRNAs by pGenesil - 1 plasmid. Methods：Specific sequences of MBP cDNA were chosen and synthesized to form complementary pairs according to shRNA expression by annealing reaction, then the obtained products containing short hairpin structure were inserted into pGenesil- 1 to construct the shRNA expression vectors of MBP, transformed into DI-15a, screened by restriction enzyme analysis and sequence analysis. Results：The recombinant plasmids expressing MBP shRNA were successfully constructed. Conclusion： We constructed recombinant expression vectors pGenesil - I - MBP - 1, pGenesil - 1 - MBP - 2, pGenesil - 1 - MBP - 3 and pGenesil - l - MBP - neg for the further study of the expression characters and mechanism of MBP gene transfer on Eukaryotic cell lines.