携带DNMT1-shRNA真核表达载体的构建及其对DNMT1表达的影响

Construction of the eukaryotic expression vector carrying double shRNA sequence targeting gene DNMT1 and its influence on expression of DNMT1.

目的构建携带针对DNA甲基转移酶1(DNMT1)mRNA的shRNA真核表达载体,检测重组质粒对DNMT1的沉默效应。方法以DNMT1mRNA序列为靶基因设计shRNA和阴性对照序列(HK),应用基因重组技术将其克隆到真核表达载体P-Gensil-1中,构建真核表达载体P-DNMT1-shRNA、P-HK。将重组质粒转染到大肠杆菌DH5α中,经筛选后对提取质粒测序鉴定。用构建的重组质粒转染T24细胞,进行RT-PCR和western blot检测,观察DNMT1mRNA和蛋白的变化。结果重组质粒经SacⅠ酶切得到大小两个基因片段,与设计相同,经测序显示插入完全正确;DNMT1mRNA在24h、48h和72h的抑制率为28.44%、52.48%、70.91%;其蛋白在24h、48h和72h的抑制率为24.27%、57.79%、69.74%。结论成功构建了P-DNMT1-shRNA真核表达载体并能有效沉默T24细胞中DNMT1mRNA和蛋白的表达。

Objective To construct and identify the eukaryotic expression vector carrying double shRNA sequence targeting DNA methltransferase 1, and to detect the silence effect of recombinant plasmid. Methods To design one strand shRNA targeting DNMT1 mRNA and a negative control（HK）, and clone them into eucaryotic expression vector P-Gensil-1 by using technology of gene recombination, and they were PshRNA-DNMT1 and P-HK. The constructed recombinant plasmids were transfected into Bacterium coli DH5α, and extracted them with Kana. The sequence of the plasmids were evaluated by electrophoresis and sequence analyzing. The silencing efficiency of DNMT1 mRNA and protein were detected by RT-PCR and western blot. Results The recombinant plasmids were separated two gene fragment by Sal Ⅰ, they are identical to the size of design, the cloning gene was detected by sequence analysis, and the results were completely right. The inhibitory rates of DNMT1 mRNA were 28. 44%, 52. 48% and 70. 91%% and those of DNMT1 protein were 24. 27%, 57. 79% and 69. 74%. Conclusion The eucaryotic expression vector can effectively silence expression of DNMT1 mRNA and protein in T24 cells.