肾癌肿瘤相关抗原G250/MN/CAⅨ基因的克隆、表达及鉴定

Study on the cloning, expression and identification of the renal cell carcinoma-associated antigen G250/MN/CA Ⅸ

目的报道G250/MN/CAⅨ基因的克隆,蛋白表达及鉴定。方法从肾癌组织中提取总RNA,采用RTPCR法扩增G250/MN/CAⅨ全长cDNA,将获得的cDNA片段插入pET22b(+)表达载体,转化BL21大肠杆菌中,以IPTG诱导进行蛋白表达,SDSPAGE凝胶电泳观察结果,实验验证。结果克隆的G250/MN/CAⅨ基因经测序证实序列正确,构建重组质粒pET22b(+)/G250并在原核系统中表达G250/MN/CAⅨ重组蛋白,该蛋白具有较好的抗原性和特异性。结论成功完成G250基因克隆,为在G250蛋白纯化基础上进行抗体制备和G250功能研究提供了实验依据。

Objective To report the cloning, expression and identification of the tumor-associated antigen G250/MN/CA Ⅸ. Methods The total RNA was extracted from renal cell carcinoma tissue sam- pies from 54 male patients. Gone fragments encoding G250 was obtained by RT-PCR, and was cloned into prekaryotic expression vector pET22b（ ＋ ） and expressed in E. coil BL21 （DE3） ;and the results were examined by SDS-PAGE gel electrophoresis. The recombinant protein was studied by Western blot test. Results DNA sequence analysis showed that the obtained sequence was the same as that showed in GenBank. Gone of G250 was expressed in E. coli BL21 successfully. Western blot analysis showed that the recombinant protein could be specially recognized by monoclonal antibody, it had better antigenicity and specificity. Conclusions This study provides experimental basis for the purification of G250/MN/CA Ⅸ protein and the further study of G250/MN/CA Ⅸ function and preparation of antibody.