肿瘤-睾丸抗原CT45-5的表达纯化及多克隆抗体的制备

Expression and purification of cancer/testis antigen 45-5 and preparation of its polyclonal antibody

目的:原核表达、纯化肿瘤-睾丸抗原CT45-5基因,并制备多克隆抗体,以研究其在Fhit特异的信号通路中的作用。方法:设计出特异针对CT45-5的引物,通过RT-PCR扩增出CT45-5的编码基因,测序正确后插入到含GST基因的原核表达载体pGEX-KG中,以IPTG诱导表达,并经谷胱甘肽琼脂糖珠纯化融合蛋白;用纯化的蛋白免疫小鼠制备多克隆抗体,经ELISA测定抗体的效价,Western印迹鉴定抗体的特异性。结果:原核表达和纯化了CT45-5,并获得了其多克隆抗体,抗体效价达到1∶12800,Western印迹结果显示,该抗血清能够特异识别原核及真核细胞表达的CT45-5蛋白。结论:肿瘤-睾丸抗原CT45-5能够诱导小鼠产生具有较高效价和特异性的多克隆抗体,为进一步研究CT45-5的功能奠定了基础。

Objective：To express and purify cancer/testis antigen 45-5 （ CT45-5 ） in E. coli, and prepare the polyclonal antibody against CT45-5 so as to study its function in the specific Fhit signal pathways. Methods：Specific primers were used to amplify CT45-5 full length gene by RT-PCR, then the gene was inserted into the vector pGEX-KG containing GST gene. GST-CT45-5 fhsion protein was expressed in E. coli, and purified by using glutathione sepharose 4B. Then anti-CT45- 5 antiserum was prepared in mice, the titer was determined by ELISA and the specificity was identified by Western blot. Results：CT45-5 was expressed and purified, and the polyclonal antibody against CT45-5 was prepared. The titer of this anti- body was about 1：12 800, and CT45-5 expressed in prokaryotic and eukaryotic cells could be recognized by this antiserum. Conclusion： CT45-5 can induce the effective and specific polyclonal antibody in mice, which sheds light on the function of cancer/testis antigen 45-5.