靶向人Bax inhibitor-1的siRNA表达质粒的构建及鉴定

Construction and identification of siRNA expressing plasmid targeted against human Bax inhibitor-1 gene

目的:构建靶向人Bax inhibitor-1的siRNA表达质粒。方法:借助siRNA设计工具,确定Bax inhibitor-1编码序列中4个可能的干扰位点,人工合成4对正反义脱氧寡核苷酸链(B1、B2、B3、B4),定向克隆至真核表达质粒pmU6,得质粒pmU6-B1、pmU6-B2、pmU6-B3、pmU6-B4。将重组质粒转染DH5α,PCR法筛选阳性克隆,DNA测序法检测插入序列的正确性。结果与结论:PCR和DNA测序结果证实各重组质粒的插入序列完全正确。靶向人Bax inhibitor-1的siRNA表达质粒构建成功。

Aim： To construct small interfering RNA （siRNA） expressing plasmids targeted against human Bax inhibitor-1 gene. Methods：Four pairs of sense and antisense sequences of short hairpin RNA, designed using web design software, were synthesized and inserted into the expression vector pmU6 with definite direction. The positive clones were screened by PCR, and the inserted sequences in the recombinant plasmids targeted to human Bax inhibitor-1 were identificated by DNA sequencing. Results ： The siRNA sequences were successfully inserted into the expression vector pmU6. Conclusion ： siRNA recombinant eukaryotic expression plasmids targeted to human Bax inhibitor-1 gene were successfully constructed.