TRPM2蛋白在大肠杆菌的表达纯化和多克隆抗体制备  

The expression and purification of TRPM2 protein in E.coli and preparation of its polyclonal antibody

作　　者：孙宏卫[1] 罗海华[1] 王妮[1] 欧小利[1] 温晓梨[1] 姜勇[1] 梅柱中[1] 

机构地区：[1]南方医科大学病理生理教研室,广东省蛋白质组学重点实验室,广东广州510515

出　　处：《细胞与分子免疫学杂志》2013年第5期511-514,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81070955)

摘　　要：目的获得人瞬时受体电位M2(TRPM2)离子通道蛋白N末端的原核表达融合蛋白,制备兔抗人TRPM2多克隆抗体。方法采用PCR扩增编码人TRPM2蛋白N末端1～334位氨基酸残基(在人与小鼠中高度保守)的cDNA序列,将其克隆至谷胱甘肽硫转移酶(GST)融合蛋白表达质粒pGEX-4T-3上。将原核表达重组质粒转化BL2l(DE3)感受态细胞,IPTG诱导GST-TRPM2N融合蛋白的表达,并纯化获得相对分子质量(Mr)约70000的GST-TRPM2融合蛋白。将此融合蛋白与弗氏完全佐剂混合后采用经典的4次免疫法免疫新西兰大白兔制备多克隆抗体,用Western blot法对该抗体进行鉴定。结果通过PCR扩增获得编码TRPM2蛋白N末端的cDNA片段并将其定向克隆至原核表达载体pGEX-4T3中,采用IPTG诱导、蛋白质的变性与复性纯化获得融合蛋白GST-TRPM2N,将纯化的融合蛋白免疫新西兰大白兔,制备获得特异性抗TRPM2蛋白的多克隆抗体。结论成功制备了特异性抗人TRPM2蛋白的多克隆抗体。Objective To purify the prokaryotically expressed GST-TRPM2N fusion protein and prepare specific poly- clonal antibody against human transient receptor potential melastatin 2 （TRPM2） protein. Methods The DNA fragment enco- ding evolutionarily conserved N-terminus （ 1-334 amino acids） of TRPM2 was amplified by PCR. The amplicon was then sub- cloned into prokaryotic expression plasmid pGEX-4T-3 and the recombinant plasmid was transformed into BL？A （DE3） cells. The expression of GST-TRPM2N fusion protein with a molecular weight about 70 000 Da was induced with ] mmoVL IPTG and purified by GST affinity chromatography. The purified protein was mixed with complete Freund＇s adjuvant and used to immunize the New Zealand white rabbits with classical 4-injection protocol to generate specific anti-TRPM2 polyclonal antibody. The specificity and titer of the anti-TRPM2 antibody was analyzed by Western blotting. Results The cDNA fragment encoding N-terminus of human TRPM2 was amplified by PCR and directionally subcloned into pGEX-4T3 plasmid. Under the induction of IPTG, we observed the expression of GST-TRPM2N fusion protein. Polyclonal antibody against human TRPM2 protein was successfully prepared in the rabbits immunized with the purified GST-TRPM2N fusion protein. And the preliminary analysis showed that the anti-TRPM2 antibody could specifically identify transiently expressed TRPM2-EE in HEK293 cells. Conclusion The specific polyclonal antibody against human TRPM2 protein has been successfully pre- pared, which facilitates our future research on the function of TRPM2 channel.

关 键 词：TRPM2 原核表达 蛋白纯化 多克隆抗体 

分 类 号：Q786[生物学—分子生物学] R392.11[医药卫生—免疫学]