泛素特异水解酶22基因RNA干扰真核表达载体的构建及鉴定

Construction and Identification of Eukaryotic Expression Plasmid Expressing siRNA Targeting USP22 Gene

目的构建泛素特异水解酶22基因(USP22)RNA干扰(RNAi)的真核表达载体,并观察其对肝癌细胞株HepG2增殖的影响。方法根据USP22 mRNA序列,合成两条寡聚DNA片段,退火后克隆入质粒载体pMSCV/Hyg/U6。酶切和测序鉴定重组质粒pMSCV/Hyg/siUSP22。脂质体介导重组质粒转染HepG2细胞,RT-PCR和Western blot检测USP22基因mRNA和蛋白表达水平,MTT法检测细胞增殖能力。结果经酶切鉴定筛选出的重组质粒测序结果与目的序列完全一致。该重组质粒转染能降低HepG2细胞USP22 mRNA及蛋白质表达,并抑制细胞增殖。结论成功构建USP22基因RNAi真核表达载体,该载体通过下调USP22基因表达,抑制HepG2细胞的增殖能力,为进一步研究USP22的生物学功能奠定了基础。

Objective To construct eukaryotic expression plasmid expressing siRNA targeting ubiquitin specific peptidase 22 gene（USP22）, and to investigate its effect on the growth of hepatoma carcinoma cells HepG2.Methods siRNA templates were synthesized based on USP22 mRNA sequence and cloned into vector Pmscv/Hyg/U6.The resulting recombinant was identified by restriction enzyme digestion and DNA sequencing.Recombinants were than transfected into HepG2 cells mediated by liposome.The USP22 protein and mRNA in HepG2 cells were detected by western blot and RT-PCR,respectively.The cellular growth activity was evaluated with MTT assay.Results Recombinant plasmid expressing siRNA targeting USP22 was successfully constructed.The down-regulated protein and mRNA level of USP22 and decreased cellular growth in HepG2 cells transfected with recombinant plasmid were observed.Conclusion The eukaryotic expression vector for RNA interference USP22 gene is constructed successfully,which inhibits the expression of USP22 in HepG2 cells and suppresses cell proliferation.