猪溶血素突变体的构建及活性评价

Construction and activities of suilysin mutants

目的:构建无溶血活性的猪溶血素突变体,并对制备蛋白进行功能评价。方法:根据猪溶素的晶体结构,采用定点突变分别将其353位脯氨酸突变为丙氨酸、亮氨酸和缬氨酸。重组表达的突变体蛋白采用镍柱亲和层析进行柱上复性后,评价纯化蛋白的溶血活性和免疫原性。结果:获得三种猪溶素突变体,SLY(P353A),SLY(P353L)和SLY(P353V)。溶血试证明SLY(P353V)无溶血活性,蛋白质免疫印迹和动物实验显示SLY(P353V)具有免疫原性。结论:猪溶素突变体SLY(P353V)无溶血活性,有免疫原性。

AIM： To construct the suilysin mutant without hemolytic activity and evaluate its functions. METHODS： The proline in 353 site of suilysin was site-directed mutated to alanine, leucine and valine, respectively. The recombinant mutants were renaturated and purified by immobilized metal ion affinity chromatography, and the purified proteins were evaluated in the hemolytic activity and immunogenicity. RESULTS： We obtained three mutants, SLY（ P353A）, SLY （P353L） and SLY（P353V）. The SLY（P353V） mutant had non-hemolytic activity. Westem blotting and animal experiments showed that SLY（P353V） mutant still had immunoge- nicity. CONCLUSION： Suilysin mutant SLY（P353V） has no hemolytic activity but remains immunogenicity.