靶基因Bi-1RNAi重组慢病毒载体的构建与鉴定

Construction and identification of a recombinant lentiviral vector of RNA interference of human Bax inhibitor-1 gene

目的构建靶向人Bax Inhibitor-1(Bi-1)基因的shRNA慢病毒重组载体。方法借助siRNA设计工具,并结合文献资料选取Bi-1基因编码序列中有效的干扰靶点,根据连接载体中的酶切位点自行设计并人工合成干扰靶点的一对反义脱氧寡核苷酸链,先定向克隆到pU6载体(pU6-Bi-1-shRNA),再经双酶切后克隆到慢病毒包装质粒LunIG,得到靶向Bi-1基因沉默的慢病毒包装重组质粒LunIG-Bi-1。重组质粒经PCR法进行初筛后,再通过双酶切电泳和目的基因沉默效果的检测保证插入序列的正确性。结果靶向沉默Bi-1基因的shRNA序列成功插入到慢病毒包装质粒LunIG中。结论靶向Bi-1基因沉默的慢病毒shRNA包装重组质粒构建成功。

Objective To construct a recombinant lentiviral vector of RNAinterference of human Bax inhibitor-1 gene （Bi-1）. Methods An effective interference target of Bi-1 gene was chosen by siRNAdesign software and references, and then one pair of sense and antisense sequences of short hairpin RNAwere designed and synthesized according to the enzyme site of plasmid. The short hairpin RNA was subcloned into lentiviral packaging plasmid LunIG after cloned into the expression vector pU6. The inserted sequences of the recombinant, named LunIG-Bi-1, were identified by PCR, double enzyme digestion and silent effect of target gene. Results The shRNA sequences targeted to silence Bi-1 gene were successfully inserted into the lentiviral packaging plasmid LunIG. Conclusion shRNAlentiviral recombinant vector targeted to Bi-1 gene can be successfully constructed.