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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, protein expression and identification of G250 / MN / CAⅨ gene. Methods Total RNA was extracted from human renal cell carcinoma (RCC). The full length cDNA of G250 / MN / CAIX was amplified by RTPCR. The cDNA fragment was inserted into pET22b (+) expression vector and transformed into BL21 E.coli for IPTG induction. SDSPAGE gel electrophoresis observations, experimental verification. Results The cloned G250 / MN / CAⅨ gene was verified by sequencing. The recombinant plasmid pET22b (+) / G250 was constructed and the recombinant protein of G250 / MN / CAIX was expressed in prokaryotic system. The recombinant protein has good antigenicity and specificity. Conclusion The successful cloning of G250 gene provides an experimental basis for antibody preparation and G250 functional study on the basis of G250 protein purification.