大鼠野生型TRAF6基因和TRAF6 shRNA表达质粒的构建及鉴定  

Construction and identification of TRAF6 gene and its shRNA expression vector

作　　者：邱文[1] 单锴[1] 庞蓉蓉[1] 季明德[1] 王迎伟[1] 

机构地区：[1]南京医科大学微生物与免疫学系,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2011年第10期1407-1411,1435,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金资助项目(31000396;81072402);江苏省高校自然科学研究(10KJB310006);南京医科大学科技发展基金面上项目(09NJMUM003);南京医科大学基础医学院青年教师培养基金(09JC007)资助

摘　　要：目的:构建大鼠野生型肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6,TRAF6)基因及其特异性短发卡状小干涉RNA(shRNA)真核表达质粒,并观察其在大鼠肾小球系膜细胞(GMC)中过表达和沉默TRAF6基因的情况。方法:用DNA重组技术将针对大鼠TRAF6基因的CDS区序列(加HA标签)和针对其不同位点所设计的3种shRNA序列分别克隆到pcDNA3.1及pGenesil-1/GFP真核表达质粒中。在酶切鉴定及序列测定正确后,用NeonTM电转仪,将上述质粒分别转染入培养的大鼠GMC中,然后用Western blot检查HA-TRAF6融合蛋白的表达情况,同时筛选最有效的TRAF6shRNA。结果:限制性酶切及核酸序列分析确证,上述TRAF6基因过表达和shRNA表达质粒均构建成功。Western blot显示,构建的pcDNA3.1-HA-TRAF6质粒能在大鼠GMC中表达,且TRAF6 shRNA-1具有最佳的沉默效率。结论:本实验成功构建了大鼠野生型TRAF6真核表达质粒及其特异性的shRNA表达载体,这为今后进一步研究TRAF6基因的生物学功能提供了实验基础。Objective： To construct tumor necrosis factor receptor-associated factor 6（TRAF6） and its small hair RNA（shRNA） expression vectors,and to assess their function on rat glomerular mesangial cell（GMC）.Methods： The full-length TRAF6 CDS（HA tag） or three kinds of shRNAs targeting TRAF6 gene were synthesized and cloned into eukaryotic expression plasmid pcDNA3.1 and pGenesil-1/GFP,respectively.After confirmed by restriction endonuclease digestion and nucleotide sequencing,the recombinant plasmids were transfected into cultured rat GMC through NeonTM transfection system.Western blot assay was then used to confirm the expression of HA-TRAF6 and find out the optimal shRNA against TRAF6 gene.Results： It was verified by restriction endonuclease digestion and nucleotide sequencing that the constructed eukaryotic vectors were all correct.Western blot assay showed that the plasmids of pcDNA3.1-HA-TRAF6 could express HA-TRAF6 and the TRAF6 shRNA-1（shTRAF6-1） was able to silence the target gene effectively.Conclusion： The expression plasmid of TRAF6 gene and its shRNA were constructed successfully,which provides essential experimental tools for studying biological functions of TRAF6 gene in the future.

关 键 词：肿瘤坏死因子受体相关因子6 小发夹RNA 肾小球系膜细胞 

分 类 号：Q782[生物学—分子生物学]