小鼠TRIM59蛋白多克隆抗体的制备、鉴定与初步应用

Preparation,characterization and preliminary application of polyclonal antibodies against mouse TRIM59

【目的】制备兔抗鼠三结构域蛋白59(Tripartite motif-containing 59,TRIM59)多克隆抗体。【方法】利用PCR方法得到小鼠TRIM59基因片段,将其克隆至pGEX-2T原核表达载体中,转入大肠杆菌BL21,IPTG诱导表达GST-TRIM59融合蛋白。GST-TRIM59融合蛋白经亲和层析纯化浓缩后免疫新西兰大耳白兔,制备多克隆抗体,通过ELISA测定兔抗鼠TRIM59多克隆抗体效价,用Western blot测定其特异性。用免疫组织化学方法检测TRIM59在小鼠前列腺癌组织中的表达。【结果】成功构建了pGEX-2T-TRIM59原核表达重组质粒,并诱导表达出GST-TRIM59融合蛋白,免疫新西兰大耳白兔5周后获得多克隆抗体。ELISA测定结果表明,TRIM59多克隆抗体效价为1∶106以上;Western blot分析表明,TRIM59多克隆抗体具有良好的特异性。免疫组织化学检测发现,TRIM59在小鼠前列腺癌组织细胞中高表达。【结论】成功地制备了特异性良好的兔抗鼠TRIM59多克隆抗体,其具有良好的免疫学活性,能够用于相关研究。

[Objective] The research prepared the high titer and specific rabbit anti-mouse antibody of TRIM59 （Tripartite motif containing 59）. [Method] The mouse TRIM59 gene was amplified by PCR. The target fragment digested by the enzyme was cloned into pGEX-2T,taking pGEX 2T-TRIM59 into BL21 for expression induced by IPTG. The expression product of TRIM59 was collected and purified. New Zealand＇s White rabbits were used to produce antiserum against GST TRIM59 recombinant proteins. The titer and specificity of the anti-mouse TRIM59 polyclonal antibodies were detected by ELISA and Western blot. [Result] GST TRIM59 fusion protein had been expressed successfully. 5 weeks after first injection,we got antiserum against GST TRIM59 recombinant proteins from rabbit. The titer of the anti-serum was above 1 ： 10^6 by EI.ISA, this antibody specific detected TRIM59 protein by Western blot. TRIM59 expression was high in prostate cancer cells by immunohistochemistry. [Conclusion] The rabbit anti-mouse TRIM59 polyclonal antibody is characterized with high titer and specificity, which is the foundation to study the characters and function of TRIM59.