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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 purify tumor-testis antigen CT45-5 gene by prokaryotic expression and to prepare polyclonal antibodies to study its role in Fhit-specific signaling pathway. Methods: The primers specific to CT45-5 were designed and the coding gene of CT45-5 was amplified by RT-PCR. The gene was inserted into the prokaryotic expression vector pGEX-KG containing GST gene and sequenced. Glutathione agarose beads were used to purify the fusion protein. The purified protein was used to immunize mice to prepare polyclonal antibody. The antibody titer was determined by ELISA and the specificity of the antibody was identified by Western blotting. Results: CT45-5 was prokaryotic expressed and purified, and its polyclonal antibody was obtained. The titer of antibody reached 1: 12800. The results of Western blotting showed that the antiserum could specifically recognize CT45-5 protein expressed in prokaryotic and eukaryotic cells. CONCLUSION: Tumor - testis antigen CT45-5 can induce polyclonal antibodies with high titer and specificity in mice, which lays the foundation for further study on the function of CT45-5.