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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 eukaryotic expression vectors carrying shRNA targeting DNA methyltransferase 3b (DNMT3b) mRNA and to screen out the most effective silencing plasmids from the constructed recombinant plasmids. METHODS: Three shRNA sequences were designed based on the DNMT3b mRNA target sequence. The recombinant plasmids pGensil-1-DNMT3b-shRNA1 and pGensil-1-DNMT3b-shRNA2 were cloned into eukaryotic expression vector pG-ensil- , PGensil-1-DNMT3b-shRNA3. After restriction analysis and sequencing analysis, the recombinant plasmids were respectively transfected into T 24 cells. The expression of DNMT3b mRNA and protein was detected by RT-PCR and Western-blot And to screen for the most effective interference sequence. Results: The recombinant plasmids were identified by restriction enzyme digestion and sequencing analysis showed that the insertion was correct. RT-PCR results showed that the inhibitory rates of shRNA1, shRNA2 and shRNA3 on DNMT3b mRNA were 20.44%, 79.91% and 54.48%, respectively. The results of Western blot showed that the inhibitory rates of shRNA1, shRNA2 and shRNA3 on DNMT3b protein were 17.27 %, 77.74% and 56.79% respectively. Conclusion: Plasmid pGensil-1-DNMT3b-shRNA was successfully constructed (1,2,3), and pGensil-1-DNMT3b-shRNA2 was selected as the most potent silencing plasmid.