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目的:制备胃癌相关蛋白GCRG224的多克隆抗体。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白兔,制备抗GCRG224的多克隆抗体。ELISA、Westernblot法鉴定抗体的效价及特异性。免疫组化染色观察GCRG224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16800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。免疫组化染色显示GCRG224在胃癌组织中的表达明显强于正常胃黏膜组织。结论:GCRG224多克隆抗体的成功制备,为进一步深入研究GCRG224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 polyclonal antibody against gastric cancer related protein GCRG224. Methods: Thioredoxin / GCRG224 fusion protein was expressed in Escherichia coli and polyclonal antibodies against GCRG224 were prepared by immunization of New Zealand white rabbits with the obtained protein. ELISA and Western blot to identify the titer and specificity of the antibody. Immunohistochemical staining was used to observe the expression of GCRG224 in gastric cancer and normal tissues. Results: Thioredoxin / GCRG224 fusion protein with relative molecular mass (Mr) of about 16800 was efficiently expressed in Escherichia coli and the protein product with nearly 100% purity was obtained by gel recovery. Anti-GCRG224 polyclonal antibody was prepared. The titer of the antibody was 1: 256000 by ELISA. Western blot confirmed that the antibody could specifically bind to the prokaryotic GCRG224 protein. Immunohistochemical staining showed that the expression of GCRG224 in gastric cancer tissues was significantly stronger than that in normal gastric mucosa tissues. Conclusion: The successful preparation of GCRG224 polyclonal antibody lays the foundation for further study on the biological function of GCRG224 and its role in the development of gastric cancer.