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目的:分别克隆小鼠白介素17受体A胞外段(mIL-17RAaa32-322)及小鼠IgG2AFc(mIgG2AFc)基因,构建mIL-17RAaa32-322-mIgG2AFc融合蛋白原核表达载体,并在体外表达、纯化及鉴定。方法:利用基因重组技术构建pET32a(+)-mIL-17RAaa32-322-mIgG2AFc原核表达载体,并在E.coli Rosetta中表达mIL-17RAaa32-322-mIgG2AFc融合蛋白,经镍离子螯合层析分离纯化后,用SDS-PAGE电泳和蛋白质印迹法进行鉴定。结果:成功构建pET32a(+)-mIL-17RAaa32-322-mIgG2AFc原核表达载体,获得高效表达的融合蛋白,且蛋白质印迹证实为目的蛋白。结论:获得mIL-17RAaa32-322-mIgG2AFc融合蛋白,为进一步研究其生物学功能奠定基础。
OBJECTIVE: To clone the mIL-17RAaa32-322 and mIgG2AFc genes of IL-17 receptor A and construct the prokaryotic expression vector mIL-17RAaa32-322-mIgG2AFc and express in vitro. And identification. Methods: The prokaryotic expression vector pET32a (+) - mIL-17RAaa32-322-mIgG2AFc was constructed by gene recombination technique. The mIL-17RAaa32-322-mIgG2AFc fusion protein was expressed in E.coli Rosetta and purified by nickel ion chelate chromatography After SDS-PAGE electrophoresis and Western blotting were used to identify. Results: The prokaryotic expression vector pET32a (+) - mIL-17RAaa32-322-mIgG2AFc was successfully constructed and the highly expressed fusion protein was obtained. Western blotting confirmed the target protein. Conclusion: The mIL-17RAaa32-322-mIgG2AFc fusion protein was obtained, which laid the foundation for further study of its biological function.