定点突变阻止金黄色葡萄球菌α-溶血素溶血活性  

Site-directed Mutagenesis of S. aureus α-Hemolysin for Deactivation of Hemolytic Activity

作　　者：代健[1] 樊自尧[1] 杨轩[2] 张丽萌[1] 陈战球 崔玉东[2] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319 [2]黑龙江八一农垦大学生命技术学院

出　　处：《黑龙江八一农垦大学学报》2014年第2期44-49,112,共7页journal of heilongjiang bayi agricultural university

基　　金：国家自然基金委资助项目(31072120);黑龙江省农垦总局资助项目(HNK11A-08-01-04)

摘　　要：金黄色葡萄球菌分泌的α-溶血素是导致肺炎的重要致病因子。为进一步研究金黄色葡萄球菌α-溶血素(α-hla)的分子致病机理以及对其进行免疫预防,对α-hla第35位氨基酸进行了定点突变。用聚合酶链式反应(PCR)技术,从S.aureus wood46株基因组中扩增出α-hla基因,再利用重叠延伸PCR技术,将第35位带正电荷的组氨酸(密码子为CAC)突变为非极性的亮氨酸(密码子为CTC)。hlaH35L基因测序结果显示第35位的组氨酸突变为亮氨酸,构建的重组表达质粒pET-28a-c(+)/hla和pET-28a-c(+)/hlaH35L在E.coli BL21(DE3)中得到表达,重组蛋白α-Hla及hlaH35L大小均为33.4 kDa,α-Hla引起兔红细胞溶血,hlaH35L未引起兔红细胞溶血。成功表达并获得了失去溶血毒性的重组蛋白hlaH35L。α-hemolysin of Staphylococcus aureus secretion was an important pathogenic factor of pneumonia. For further study of Staphylococcus aureus of α-hemolysin （α-hla） the molecular pathogenesis and immunization, the thirty-fifth amino acids of α-hla was applied the site-directed mutagenesis. Using polymerase chain reaction （PCR） technology,α-hla gene was amplified from S. aureus wood46 strain genome, and the thirty-fifth with a positively charged histidine （codon CAC） mutated into non-polar leucine （codon CTC） by the reused overlap extension PCR technique. The results of hlaH35L gene sequencing showed the thirty-fifth histidine mutated into leucine, and the constructed recombinant plasmid pET-28α-c （＋）/hla and pET-28α-c （＋）/hlaH35L were expressed in E.eoli BL21 （DE3）;recombinant protein α-Hla and hlaH35L sizes were 33.4kDa; α-Hla caused rabbit erythroeyte hemolysis and hlaH35L didn＇t cause rabbit erythrocyte hemolysis,which expressed and got recombinant proteins hlaH35L of lost hemolytic toxicity.

关 键 词：金黄色葡萄球菌 α-溶血素 定点诱变 原核表达 溶血活性 

分 类 号：S482[农业科学—农药学]