Sun Hongwei, Luo Haihua, Wang Ni, Ou Xiaoli, Wen Xiaoli, Jiang Yong, Mei Zhuzhong
Key Laboratory of Functional Proteomics of Guangdong Province, Department of Pathophysiology, Southern Medical University, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 May;29(5):511-4.
To purify the prokaryotically expressed GST-TRPM2N fusion protein and prepare specific polyclonal antibody against human transient receptor potential melastatin 2(TRPM2) protein.
The DNA fragment encoding evolutionarily conserved N-terminus (1-334 amino acids) of TRPM2 was amplified by PCR. The amplicon was then subcloned into prokaryotic expression plasmid pGEX-4T-3 and the recombinant plasmid was transformed into BL21 (DE3) cells. The expression of GST-TRPM2N fusion protein with a molecular weight about 70 000 Da was induced with 1 mmol/L 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.
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.
The specific polyclonal antibody against human TRPM2 protein has been successfully prepared, which facilitates our future research on the function of TRPM2 channel.
纯化原核表达的GST-TRPM2N融合蛋白，并制备抗人瞬时受体电位香草酸亚型2（TRPM2）蛋白的特异性多克隆抗体。
通过PCR扩增编码TRPM2进化保守N端（1-334个氨基酸）的DNA片段。然后将扩增产物亚克隆到原核表达质粒pGEX-4T-3中，并将重组质粒转化到BL21（DE3）细胞中。用1 mmol/L异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达分子量约为70 000 Da的GST-TRPM2N融合蛋白，并通过GST亲和层析进行纯化。将纯化的蛋白与完全弗氏佐剂混合，按照经典的四针注射方案免疫新西兰白兔，以产生特异性抗TRPM2多克隆抗体。通过蛋白质免疫印迹法分析抗TRPM2抗体的特异性和效价。
通过PCR扩增出编码人TRPM2 N端的cDNA片段，并定向亚克隆到pGEX-4T3质粒中。在IPTG诱导下，观察到GST-TRPM2N融合蛋白的表达。用纯化的GST-TRPM2N融合蛋白免疫的兔子成功制备了抗人TRPM2蛋白的多克隆抗体。初步分析表明，抗TRPM2抗体能够特异性识别在HEK293细胞中瞬时表达的TRPM2-EE。
已成功制备抗人TRPM2蛋白的特异性多克隆抗体，这将有助于我们今后对TRPM2通道功能的研究。
