猪链球菌ssnA基因缺失突变菌株的构建及毒力分析

摘要

Streptococcus suis nuclease A (ssnA) is a recently discovered deoxyribonuclease (DNase),which is one of potential virulence factors of Streptococcus suis serotype 2(SS2).To determine the effects of ssnA on virulence,the ssnA deletion mutant (ΔssnA) was constructed by using the suicide vector pSET4S.The growth and hemolytic activity of the ssnA gene mutant strain had no obvious difference compared with the wild-type strain.The ability of ΔssnA mutant to interact with human laryngeal epithelial cell (Hep-2) was evaluated and it exhibited dramatically decreased ability to adhere to and invade Hep-2 cells.For CD1 mice,the LD50 of the mutation strain was 6.17×108 cfu,whereas the LD50 of wild-type strain was 4.42×107 cfu.This mutation was found to exhibit significant attenuation of virulence when evaluated in CD1 mice,suggesting ssnA plays a critical role in the pathogenesis of SS2,the mechanism of this protein is valuable for further analysis.%分泌型核酸酶基因ssnA是近年来研究发现的一种脱氧核糖核酸酶,被认为是一种猪链球菌2型(SS2)潜在的毒力因子.为了分析ssnA基因对于SS2毒力的影响,通过体外构建具有同源臂的重组自杀性质粒,电转化进入 SS2,经同源重组构建了SS2流行毒力株GD01的ssnA基因缺失突变菌株,并通过比较分析了野生菌株与缺失突变菌株的生长特性及溶血活性等生物学特性.结果显示,GD01株与ΔssnA突变株的生长速度及溶血活性没有明显差异;ssnA基因缺失后SS2对Hep-2细胞的黏附及入侵能力则明显下降.小鼠毒力试验结果显示,ssnA基因缺失突变菌株对于CD1小鼠LD50是6.17×108 cfu,而SS2野生菌株的LD50 是4.42×107 cfu,缺失突变株的LD50约是野生株的14倍,表明ssnA的缺失对SS2毒力产生明显影响,提示ssnA基因在其致病过程中具有重要作用,其具体作用机制需进一步分析.