DNMT3a基因shRNA质粒表达载体的构建及其沉默作用

Construction of the eukaryotic expression vector of DNMT3a shRNA and verification of its effect on silencing

目的以DNMT3a mRNA保守序列为靶基因设计针对DNMT3a mRNA的siRNA序列和无效干扰对照序列(HK),并利用基因重组技术将其克隆到真核表达载体p Gensil-1中,构建p Genesil-1-DNMT3a-shRNA、p Genesil-1-HK-shRNA重组质粒。方法应用转化技术将重组质粒转化到大肠杆菌DH5a中,通过卡那霉素抗性筛选出阳性菌落后提取质粒进行酶切和测序鉴定,应用质粒转染技术转染耐顺铂肺腺癌A549细胞(A549-DDP),利用RT-PCR技术检测转染重组质粒24、48、72 h后DNMT3a的沉默效率。结果测序结果与合成序列一致,重组质粒不能够被PstⅠ切开,转染p Genesil-1-DNMT3a-shRNA能够明显抑制DNMT3a的表达,抑制率分别达到25.5%,56.2%,63.4%,且呈明显时间依赖性,而p Genesil-1-HKshRNA无明显抑制作用。结论成功构建了DNMT3a基因shRNA及HK质粒真核表达载体,为进一步实验研究奠定了基础。

Objective To construct the eukaryotic expression vector carrying double shRNA sequence targeting conserved domain of DNMT3 a mRNA and negative control( HK) in p Gensil-1 by using technology of gene recombination. Methods The recombinant plasmids were transformed into competent Escherichia coli DH5α. The positive clone E. coli was screened and enriched by kanamycin( 50 μg / ml),and then the recombinant plasmids were extracted from the DH5α and evaluated by restriction enzymes and sequence analysis. Cisplatin-resistant human lung adenocarcinoma A549 cells( A549-DDP) were chosen and transfected with the recombinant plasmids,semi-quantitative RT-PCR was performed to confirm the inhibitory rates of DNMT3 a mRNA before and after plasmid transfecting in 24,48,72 h. Results Sequence analysis showed the right sequence and the recombinant plasmids couldn’t be digested by PstⅠ,and p Genesil-1-DNMT3a-shRNA transfected could significantly decrease the DNMT3 a mRNA with the inhibitory rates of 25. 5%,56. 2%,63. 4%,respectively,and the effect was dependent on the time duration. However,p Genesil-1-HK-shRNA barely affected the expression of DNMT3 a mRNA. Conclusions The eukaryotic expression vectors are successfully constructed,which laids a foundation of further research.