双点突变对猪链球菌2型溶血素活性的影响
Influence of Double Positions Mutant of Streptococcus suis Serotype 2 Suilysin to Its Hemolytic Activity
  
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中文摘要:
      猪链球菌2型作为一种人兽共患病病原,日益受到关注。而溶血素是其分泌的外毒素,是公认的重要毒力因子之一,具有良好的免疫原性。本试验通过PCR方法获得猪链球菌2型野生型溶血素基因sly和463、464双点突变的溶血素突变体基因slym。将sly和slym克隆至表达载体,构建了2个重组载体并在大肠杆菌中表达了野生型溶血素rSLY和突变型溶血素rSLYm。经过蛋白杂交试验,证明表达的rSLY和rSLYm与提取的猪链球菌的天然野生型SLY分子量完全一致。以提取的野生型SLY为对照,通过溶血试验证明,突变型溶血素rSLYm失去了溶血活性;通过接种PK15、RK13、SUVEC细胞单层,证明突变型溶血素rSLYm失去细胞毒性;通过小鼠试验,证明突变型SLYm对小鼠没有毒力。本试验通过溶血试验、细胞接种和小鼠实验,证明双点突变灭活了野生型溶血素的溶血活性、细胞毒性和小鼠毒力。该溶血素突变体经免疫实验证实后,可作为猪链球菌2型亚单位疫苗的候选抗原。
英文摘要:
      Streptococcus suis serotype 2(SS2)is a zoonosis pathogen and of more and more concern. Suilysin(SLY)is a well-known antigenic virulence factor that secreted by SS2 to the extracellular environments,which has good immunogenicity. In this study,the wild type sly gene and mutant slym gene which has position 463 and 464 mutations were amplified by PCR. Then these genes were inserted into expression plasmids and two E. coli expression systems were constructed respectively. These two systems might express wild type rSLY or rSLYm. The proteins hybrid results showed that rSLY and rSLYm had same molecular weight with the natural wild type SLY. Compared with rSLY,Hemolytic test indicated rSLYm lost its hemolytic ability. Cytotoxicity test with K15,RK13 and SUVEC monolayer cell showed that rSLYm also lost cytotoxicity. Based on mice test,it was failed to prove that rSLYm could induce mouse death. With these Hemolytic test,Cytotoxicity test and animal test,it was demonstrated that mutant rSLYm could be considered as a candidate subunit vaccine antigen of SS2.
作者单位
刘爽,张秀娟,张兴进,宫枫举,潘子豪,马喆,孙学强  
中文关键词:  猪链球菌2型  溶血素  突变  溶血活性  细胞毒性
英文关键词:Streptococcus suis serotype 2  Suilysin  mutation  hemolyticity  cytotoxicity
基金项目:科技部科技基础性工作专项(2012FY111000)
DOI:10.3969/j.issn.1005-944X.2017.09.024
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