人UbcH10及其siRNA真核表达载体的构建与表达  

Study on the Construction of Eukaryotic Expression Plasmids of UbcH10 and Its siRNA Segment with Effective Expression in LOVO Cells

作　　者：陈世敏[1,2] 徐春晓[2] 刘斌[2] 辛家璇[2] 刘贤锡[2] 

机构地区：[1]济南军区总医院实验诊断科,250031 [2]山东大学医学院生物化学与分子生物学研究所,250012

出　　处：《放射免疫学杂志》2009年第4期397-399,共3页Journal of Radioimmanology

摘　　要：目的:构建以人泛素结合酶UbcH10基因为基础的真核表达质粒pcDNA3.1(UbcH10)及其siRNA表达载体pGPU6/GFP/Neo(siRNA-UbcH10),并在结肠癌细胞LOVO中表达与鉴定。方法:提取组织总RNA,通过RT-PCR扩增出UbcH10基因,克隆到真核表达载体pcDNA3.1的多克隆位点中,构建UbcH10的真核表达载体。通过siRNA设计软件选取UbcH10基因的siRNA片段合成构建表达载体pGPU6/GFP/Neo(siRNA-UbcH10)。然后采用脂质体转染法将其分别转染LOVO细胞,用RT-PCR在RNA水平和Western Blot在蛋白水平检测其在真核细胞中的表达。结果:测序结果显示克隆UbcH10基因片段序列完全正确;重组质粒转染LO-VO细胞后,检测到pcDNA3.1(UbcH10)转染组UbcH10表达增强,而pGPU6/GFP/Neo(siRNA-UbcH10)转染组UbcH10表达减弱。结论:成功构建真核表达质粒pcDNA3.1(UbcH10)及其siRNA表达载体pGPU6/GFP/Neo(siRNA-UbcH10),并在真核细胞中能有效表达,为今后进一步研究UbcH10的功能和作用机制奠定了基础。Objective To construct eukaryotic expression plasmid pcDNA3.1（UbcH10） and pGPU6/GFP/Neo（siRNA-UbcH10）,and study their expressions in human colon cancer cell line LOVO.Methods The UbcH10 cDNA was obtained with RT-PCR from total RNA extracted from the tissue,furthur cloned into pcDNA3.1 to construct the recombinant expression plasmid pcDNA3.1（UbcH10）.The siRNA-UbcH10 segment was synthesized（based on siRNA designing software） and the expression plasmid pGPU6/GFP/Neo（siRNA-UbcH10） was constructed.Both the expression plasmids were transfected into human colon cancer cell line LOVO with the liposome lipofectamine 2000.Finally,the UbcH10 gene expression of protein was detected with RT-PCR on mRNA level and Western Blot on protein level.Results The length and sequence of the cloned UbcH10 segment was correct.The UbcH10 expression in the group of pcDNA3.1（UbcH10） increased obviously but in the group of pGPU6/GFP/Neo（siRNA-UbcH10） reduced obviously in transfected LOVO cells.Conclusion The eukaryotic expression plasmids pcDNA3.1（UbcH10）and pGPU6/GFP/Neo（siRNA-UbcH10） were successfully constructed and effectively expressed in LOVO cells.This will facilitate furthur study on UbcH10 function.

关 键 词：UBCH10 真核表达 SIRNA 

分 类 号：R73-362[医药卫生—肿瘤] R512.62[医药卫生—临床医学]