携带DNMT3b-shRNA干扰序列真核表达载体的构建及筛选

Construction and Selection of Eukaryotic Expression Vectors Carrying shRNA Targeting DNMT3b

目的:构建携带针对靶基因DNA甲基转移酶3b(DNA methyltransferase 3b,DNMT3b)mRNA的shRNA真核表达载体,并从构建成功的重组质粒中筛选出沉默效应最强的干扰质粒。方法:以基因DNMT3bmRNA为靶序列设计三条shRNA序列,应用基因重组技术将其克隆到真核表达载体pG-ensil-1中,构建重组质粒pGensil-1-DNMT3b-shRNA1、pGensil-1-DNMT3b-shRNA2、pGensil-1-DNMT3b-shRNA3;经酶切鉴定和测序分析后,将重组质粒分别转染T_(24)细胞中,应用RT-PCR和Western-blot检测各组质粒对DNMT3bmRNA和蛋白的表达抑制情况,并筛选最有效的干扰序列。结果:各重组质粒经酶切鉴定和测序分析显示插入完全正确。RT-PCR结果经图像分析,shRNA1、shRNA2和shRNA3对DNMT3bmRNA的抑制率分别为20.44%、79.91%和54.48%;Western blot结果经图像分析,shRNA1、shRNA2和shRNA3对DNMT3b蛋白的抑制率分别为17.27%、77.74%和56.79%。结论:成功构建了质粒pGensil-1-DNMT3b-shRNA(1,2,3),并筛选出pGensil-1-DNMT3b-shRNA2为沉默效应最强的质粒。

Objective：To construct three eukaryotic expression vectors carrying shRNA targeting DNA methyltransferase 3b （DNMT3b）, and select the most effective vector for gene silencing. Methods：Three pairs of shRNA sequence targeting DNMT3b mRNA were designed and cloned into pGensil 1 vectors by using technique of gene recombination, and recombinant plasmids pGensil-1-DNMT3b-shRNA1, pGensil-1 DNMT3b-shRNA2, pGensil-1-DNMT3b-shRNA3 were constructed. After restriction enzyme digestion and sequencing analysis, the recombinant plasmid were transfected into T 24 cells. The expressions of DNMT3b mRNA and protein were detected by RT-PCR and western blot and the most effective vector was selected. Results： The recombinant plasmids were separated into two gene fragment by restriction enzyme. The sequences of the vectors were completely confirmed by sequencing. The mRNA inhibition of DNMT3b mRNA of three groups, pGensil-l-DNMT3b shRNA（1, 2,3）, were 20.44%, 79.91%, and 54.48%, respectively; and the protein inhibition of DNMT3b protein of them were 17.27%, 77.74%, and 56.79% , respectively by using imaging analysis. Conclusions：Three eukaryotic ex pression vectors pGensil 1-DNMT3b-shRNA（1,2,3） were constructed successfully, and pGensil-1-DNMT3b shR NA2 was selected for the further study as the most effective plasmid of DNMT3b silencing.