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目的:制备GCRG213单克隆抗体(mAb)并进行初步鉴定。方法:在大肠杆菌中表达HIS-GCRG213融合蛋白,并以所获蛋白作为免疫原制备鼠mAb。采用ELISA、Westernblot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:HIS-GCRG213融合蛋白在大肠杆菌中获得高效表达,经常规的细胞融合和筛选获得2株可稳定分泌抗GCRG213的杂交瘤细胞株。ELISA法检测腹水的效价可达到1∶106,Western blot证实该抗体可与重组HIS-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:成功地制备出2株抗GCRG213的mAb,为进一步研究GCRG213的生物学功能提供了有效的工具。
OBJECTIVE: To prepare monoclonal antibody (mAb) GCRG213 and conduct preliminary identification. Methods: The HIS-GCRG213 fusion protein was expressed in E. coli and the murine mAb was prepared using the obtained protein as an immunogen. The antibody titer and specificity were identified by ELISA and Western blot. Immunohistochemical staining was used to observe the expression of GCRG213 in gastric cancer and normal tissues. Results: The HIS-GCRG213 fusion protein was highly expressed in E. coli. Two hybridoma cell lines stably secreting GCRG213 were obtained by conventional cell fusion and screening. Ascites titer was 1:106 by ELISA. Western blot showed that the antibody could specifically bind to recombinant HIS-GCRG213 protein. Immunohistochemical staining showed that the expression of GCRG213 in gastric cancer tissues was significantly higher than that of normal gastric mucosa. Conclusion: Two mAbs against GCRG213 were successfully prepared, which provided an effective tool for further study on the biological function of GCRG213.