shRNA表达载体的改建及鉴定

基础医学与临床 ›› 2011, Vol. 31 ›› Issue (1): 78-83.

shRNA表达载体的改建及鉴定

陈娴,谢渊,赵艳,周建奖

贵阳医学院

收稿日期:2009-09-07 修回日期:2010-05-04 出版日期:2011-01-05 发布日期:2011-01-05
通讯作者: 周建奖

Reconstruction and identification of small hairpin RNA expression vector

CHEN Xian ¹,XIE Yuan ²,ZHAO Yan ²,ZHOU Jian-jiang ¹

1. Guiyang Medical College2.

Received:2009-09-07 Revised:2010-05-04 Online:2011-01-05 Published:2011-01-05
Contact: ZHOU Jian-jiang

摘要/Abstract

摘要： 目的通过改建pSilencer3.1-H1载体快速有效筛选重组shRNA表达载体，并可使线性化载体环化，长期保存。方法制备含有单一限制性内切酶NotⅠ识别序列的双链DNA插入片段，与BamHⅠ和HindⅢ酶切线性化的shRNA表达载体pSilencer3.1-H1连接，构建载体pSilencer3.1-H1/NotⅠ，再用BamHⅠ和HindⅢ双酶切pSilencer3.1-H1/NotⅠ，将含靶向目的基因JAK2 siRNA表达框的DNA模板与其连接，构建pSilencer3.1-H1/JAK2 的shRNA表达载体，提取质粒DNA，用NotⅠ进行单酶切，快速选择阳性克隆，选取不能被NotⅠ切开的质粒进行测序鉴定。随后将pSilencer3.1-H1/JAK2转染胃癌细胞株，用Western blot 检测JAK2蛋白的表达。结果通过测序证实pSilencer3.1-H1 /NotⅠ和含JAK2 siRNA表达框的表达载体成功构建，将其转染胃癌细胞株AGS后，抑制了JAK2蛋白的表达。结论通过对pSilencer3.1-H1表达载体的改建可以快速有效的筛选shRNA表达载体。

关键词: RNA干扰小发卡RNA基因沉默表达载体

Abstract: Objective To develop an effective and rapid method for screening recombinant hairpin RNA expression plasmids through reconstructing pSilencer3.1 -H1 vector. Methods The double-strand DNA fragement containing a NotⅠrestriction endonuclease site, the flanking sequences of which were not complementary, was annealed and ligated into small hairpin RNA (shRNA) expression vector pSilencer3.1-H1 to construct the pSilencer3.1-H1/NotⅠvector. With BamHⅠand Hind III, the pSilencer3.1-H1/NotⅠwas digested and ligated with siRNA expression cassette targeting JAK2 gene. The plasmid DNA of the positive clones was extracted and digested with NotⅠ. The un-digested vector was selected to identify by sequence analysis to construct the vector pSilencer3.1-H1/JAK2. After transfection of pSilencer3.1-H1/JAK2 into gastric cancer AGS cells, the JAK2 protein was detected by Western blotting. Results The sequencing results showed that pSilencer3.1-H1/NotⅠand pSilencer3.1-H1/JAK2 vectors were successfully constructed. The JAK2 protein was inhibited in transfected AGS cells. Conclusion The reconstructed pSilencer3.1-H1/ NotⅠvector could rapidly and efficiently screen shRNA expression vecteors.

陈娴;谢渊;赵艳;周建奖. shRNA表达载体的改建及鉴定[J]. 基础医学与临床, 2011, 31(1): 78-83.

CHEN Xian ;XIE Yuan ;ZHAO Yan ;ZHOU Jian-jiang . Reconstruction and identification of small hairpin RNA expression vector[J]. Basic & Clinical Medicine, 2011, 31(1): 78-83.