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pSilenc-er3.1-H1/NotⅠ,将含靶向目的基因JAK2siRNA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转染胃癌细胞系,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JAK2siRNA表达框的表达载体成功构建,将其转染胃癌细胞系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地筛选shRNA表达载体。
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 fragment containing a Not Ⅰrestriction endonuclease site was annealed and ligated into small hairpin RNA（shRNA） expression vector pSilencer3.1-H1 to construct the pSilencer3.1-H1/Not Ⅰvector.With BamH Ⅰand Hind Ⅲ,the pSilencer3.1-H1/Not Ⅰwas digested and ligated with siRNA expression cassette targeting JAK2 gene.The plasmid DNA of the positive clones was extracted and digested with Not Ⅰ.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 blot.Results 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.
出处
《基础医学与临床》
CSCD
北大核心
2011年第1期78-83,共6页
Basic and Clinical Medicine
基金
国家自然科学基金(30560038)
贵州省国际科技合作重点项目计划(黔科合外G字
700119)
