真核细胞人突变CD59的纯化及初步鉴定

目的:获得人突变CD59(hmCD59)蛋白,为后续研究提供必要的材料。方法:运用脂质体介导法,将含有hmCD59全长cDNA序列的重组pALTER质粒与pcDNA3质粒共转染CHO细胞,以G418筛选阳性克隆,以免疫荧光技术和SDS-PAGE检测hmCD59的表达,表达产物经Anti-FLAGM2亲和凝胶纯化后,以SDS-PAGE、Western印迹和ELISA对纯化产物进行鉴定。结果:hmCD59蛋白在转染后的CHO细胞表面稳定表达。SDS-PAGE结果表明,纯化的hmCD59的相对分子质量同预期结果一致。Western印迹和ELISA证实,纯化的hmCD59蛋白具有与抗CD59抗体结合的活性。结论:获得了电泳纯的hmCD59蛋白,为进一步对其进行抗体制备、功能研究及临床应用奠定了基础。

Purification and Identification of Eukaryotic Human Mutant CD59

Objective:To obtain human mutant CD59(hmCD59)protein,so as to facilitate further research.Methods:The recombinant pALTER plasmid containing hmCD59 cDNA was transfected into CHO cells with pcDNA3 by lipofectamine.Stably transfected clones were then selected by G418.Expression of hmCD59 on CHO cells was detected by fluorescent antibody technique and SDS-PAGE.The recombinant protein was purified by Anti-FLAG M2 affinity gel chromatography and identified by SDS-PAGE,Western-blot and ELISA.Results:The hmCD59 was expressed on transfected CHO cells' surface.SDS-PAGE analysis showed that the relative molecular mass of hmCD59 protein obtained was correct as predicted.Western-blot and ELISA data revealed that it could react with anti-CD59 antibodies.Conclusion:The purified hmCD59 protein has been obtained successfully,which lays the foundation for preparing antibody,further functional study and clinic application of hmCD59.