抗人胃癌相关蛋白GCRG224多克隆抗体的制备及鉴定

Preparation and identification of polyclonal antibody against gastric cancer-related protein GCRG224

目的制备胃癌相关蛋白GCRG224的多克隆抗体,为进一步深入研究其生物学功能及其在胃癌发生发展中的作用奠定基础。方法在大肠杆菌中表达Thioredoxin/GCRG224融合蛋白,并以所获蛋白免疫新西兰兔,制备抗GCRG224的多克隆抗体。分别用ELISA、Westernblot法鉴定抗体的效价及特异性。结果在大肠杆菌中高效表达出相对分子量约为16.8kD的Thioredoxin/GCRG224融合蛋白,经凝胶回收法得到纯度近100%的蛋白产品。制备了抗GCRG224多克隆抗体。ELISA法检测抗体的效价达到1∶256000,Westernblot证实该抗体可与原核表达的GCRG224蛋白特异性结合。结论成功地制备了胃癌相关蛋白GCRG224多克隆抗体。

Objective To prepare a polyclonal antibody against gastric cancer-related protein GCRG224. Methods The thioredoxin/ GCRG224 fusion protein was expressed in E. coll. A polyclonal antibody against GCRG224 was obtained by immunizing a rabbit with the purified GCRG224 protein. The titer and specificity of the antibody were determined by ELISA and Western-blot, respectively. Results The thioredoxin/GCRG224 fusion protein with relative molecular mass of 16. 8kD was over-expressed in E. coll. The purity of expressed products directly purified from a denaturing polyacrylamide gel was about 100%. The polyclonal antibody against GCRG224 was obtained. The ELISA titer of antiserum against GCRG224 was about 1：256 000. Western blot analysis showed that the antiserum could bind to the expressed fusion protein specificaUy. Conclusion The polyclonal antibody against GCRG224 has been successfully prepared, which lays the foundation for further study on the biological function and the possible role of the GCRG224 in the development of gastric carcinoma.