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构建胱抑素C原核表达载体p Cold1-cysc,p ET28a-cysc,p ET32a-cysc和p ET41a-cysc及相应的BL21(DE3)表达菌株,最终得到重组人胱抑素C蛋白。应用基因工程手段,原核表达,亲和层析及透析复性的方法对目的蛋白进行表达,纯化和复性。结果:经分析后,选取表达量较大的菌株BL21(DE3)p ET32a-cysc和BL21(DE3)p Cold1-cysc,对目的蛋白进行提取及纯化,对纯化后的可溶及包涵体形式的重组胱抑素C进行透析复性及免疫活性的检测。Western blotting及ELISA结果显示,携带有不同融合标签的可溶性重组胱抑素C或包涵体在透析复性后具有不同的免疫活性。结论:在大肠杆菌中成功表达并得到人胱抑素C的蛋白,复性后得到了重组胱抑素C的活性形式。
Recombinant human cystatin C protein was obtained by constructing the prokaryotic expression vector pCold1-cysc, pET28a-cysc, p ET32a-cysc and pET41a-cysc of cystatin C and the corresponding BL21 (DE3) expressing strain. The target protein was expressed, purified and refolded by genetic engineering, prokaryotic expression, affinity chromatography and dialysis refolding. Results: After the analysis, we selected the strains BL21 (DE3) p ET32a-cysc and BL21 (DE3) p Cold1-cysc which had higher expression level to extract and purify the target protein. The purified soluble and inclusion bodies Recombinant cystatin C for dialytic refolding and immunocompetence detection. Western blotting and ELISA showed that soluble recombinant cystatin C or inclusion bodies carrying different fusion tags had different immunoreactivities after dialysis refolding. Conclusion: The recombinant protein of cystatin C was successfully expressed in E. coli and the recombinant form of recombinant cystatin C was obtained after renaturation.