抗人胃癌相关蛋白GCRG213多克隆抗体的制备鉴定及初步应用

Preparation, identification and preliminary application of polyclonal antibody against gastric cancer-related protein GCRG213

目的制备胃癌相关蛋白GCRG213的多克隆抗体。方法在大肠杆菌中表达Thioredoxin／GCRG213融合蛋白，并以所获蛋白免疫新西兰白兔，制备兔抗GCRG213的抗体。酶联免疫吸附测定（ELISA）、Western blot法鉴定抗体的效价及特异性。免疫组化染色观察GCRG213在胃癌和正常组织中的表达。结果在大肠杆菌中高效表达出相对分子质量（Mr）约为29400的thioredoxin／GCRG213融合蛋白，经凝胶回收法得到纯度近100％的蛋白产品。制备兔抗GCRG213抗体。ELISA法检测抗体的效价达到1：256000，Western blot证实该抗体可与原核表达的GCRG213蛋白特异性结合。免疫组化染色显示GCRG213在胃癌组织中的表达明显强于正常胃黏膜组织。结论兔抗GCRG213抗体的成功制备，为进一步研究GCRG213的生物学功能及其在胃癌发生发展中的作用奠定了基础。

Objective To prepare the polyclonal antibody against gastric cancer related protein GCRG213. Methods The thioredoxin/GCRG213 fusion protein was expressed in E. coll. The polyclonal antibody against GCRG213 was obtained by immunizing a rabbit with the purified GCRG213 protein. The titer and specificity of the antibody were determined by ELISA and Western blot, respectively. The expressions of GCRG213 on paraffin-embedded tissue sections from normal gastric mucosal tissues and advanced gastric cancer were determined using immunohistoehemistry technique. Results The thioredoxin/GCRG213 fusion protein with relative molecular mass （Mr） of 29 400 was over-expressed in E. coli. The purity of the expressed product directly purified from a denaturing polyacrylamide gel was about 100 %. The rabbit antibody against GCRG213 was obtained. The ELISA titer of antiserum against GCRG213 was about 1 ： 256 000. Western blot analysis showed that the antiserum could bind to the expressed fusion protein specifically. The immunohistochemistry showed that the expression of GCRG213 was higher in gastric cancer tissues than in normal ones. Conclusions The rabbit antibody against GCRG213 has been successfully prepared, which may establish a foundation of further studying the biological function and the possible role of the GCRG213 in the development of gastric carcinoma.