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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。
Objective: To construct hemolysin-free mutants without hemolytic activity and to evaluate the function of the prepared proteins. Methods: According to the crystal structure of porcine lysine, the 353-position proline was mutated to alanine, leucine and valine respectively by site-directed mutagenesis. Recombinantly expressed mutant protein was purified on nickel column affinity chromatography column to evaluate the hemolytic activity and immunogenicity of the purified protein. RESULTS: Three pig lysine mutants were obtained, SLY (P353A), SLY (P353L) and SLY (P353V). Hemolysis showed that SLY (P353V) had no hemolytic activity. Western blotting and animal experiments showed that SLY (P353V) was immunogenic. Conclusion: The pig lysine mutant SLY (P353V) has no hemolytic activity and immunogenicity.