人TRAF3IP3基因的克隆及真核表达  

Cloning and eukaryotic expression of human TRAF3IP3 gene

作　　者：王沂[1] 张秀军[2] 刘青[2] 余源[2] 慈雅丽[2] 王洋[2] 

机构地区：[1]河北联合大学附属医院检验科 [2]河北联合大学生命科学学院,河北唐山063000

出　　处：《细胞与分子免疫学杂志》2013年第5期465-468,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81072093);河北省青年科学基金(C2012401039)

摘　　要：目的构建人肿瘤坏死因子受体相关因子3相互作用蛋白3(TRAF3IP3)基因真核表达载体,并进行表达检测及鉴定。方法 RT-PCR扩增获得TRAF3IP3开放读码框区基因,双酶切连入真核表达载体pIRES2-EGFP,双酶切和测序鉴定阳性克隆;重组质粒磷酸钙法转染HEK293细胞,荧光显微镜观察绿色荧光蛋白的表达,实时定量PCR和Western blot法鉴定TRAF3IP3基因的表达情况。结果经限制性内切酶酶切鉴定和测序证实,成功构建了TRAF3IP3真核表达质粒,荧光显微镜观察可见转染了重组质粒的HEK293细胞有绿色荧光蛋白的表达,实时定量PCR和Western blot结果证实TRAF3IP3蛋白高水平表达。结论成功构建了pIRES-EGFP-TRAF3IP3真核表达质粒,为TRAF3IP3基因功能的研究奠定了基础。Objective To construct an eukaryotic expression plasmid of human TNF receptor-associated factor 3 in ter- acting protein 3（TRAF31P3） gene and identify its expression in HEK293 cells. Methods Human TRAF31P3 cDNA was ampli- fied by RT-PCR from bone marrow mononuclear cells. After digested by restriction enzymes Xho I and Sal I, the complete open reading frame of TRAF31P3 gene was inserted into plRES2-EGFP eukaryotic expression vector with a Flag tag at the N- terminus. The recombinant plasmid was identified by double restriction enzyme digestion and sequencing analysis. The con- structed TRAF31P3 eukaryotic expression plasmid was transfected into HEK293 cells by calcium phosphate precipitation meth- od. The expression of green fluorescence protein was observed by fluorescence microscopy. Real-time PCR and Western blotting were performed to detect the expression of Flag-TRAF31P3 fusion protein. Results Double restriction enzyme diges- tion and sequencing analysis revealed that TRAF31P3 eukaryotic expression plasmid was constructed successfully. Green fluorescence was detected in transfected HEK293 cells. Real-time PCR showed TRAF31P3 mRNA was expressed at a high level. The approximate 62 kD Flag-TRAF31P3 fusion protein was found by Western blotting. Conclusion Human TRAF31P3 eukaryotic expression plasmid plRES2-EGFP-TRAF31P3 has been constructed successfully, which provides a foundation for the gene function research of TRAF31P3.

关 键 词：TRAF3IP3 真核表达 转染 HEK293细胞 

分 类 号：R392.33[医药卫生—免疫学] Q343.1[医药卫生—基础医学]