猪链球菌2型srtA基因缺失菌株的构建及生物学特性

Construction and characteristic of srtA gene mutant strain of Streptococcus suis 2

扩增了srtA基因的上、下游同源臂P1和P2及其全长序列,利用温度敏感型"自杀性"质粒pSET4s构建了重组质粒pSET4s-P1-P2,并将该质粒电转化入野生菌株SS2(SC21)中,通过抗生素和温度双重筛选,得到srtA基因缺失菌株,命名SC211。同时,将srtA基因定向插入穿梭质粒pAT18,构建穿梭质粒pAT18-srtA,并将该质粒电转入srtA基因缺失菌株SC211中,通过抗生素筛选,得到质粒介导的srtA基因互补菌株SC212。通过PCR、South-ern blotting等对缺失突变菌株和互补菌株进行了鉴定。对基因缺失突变菌株和回复菌株传代培养遗传稳定性试验结果显示,缺失突变菌株和互补菌株能够稳定遗传。比较了基因缺失突变菌株、互补菌株及野生菌株的生长特性、溶血活性、细胞粘附特性,结果表明,3个菌株的生长速度、溶血活性没有明显差异。基因缺失后SS2对Hep2细胞的粘附能力明显下降,只有野毒菌株的49%,互补菌株粘附能力几乎达到野毒菌株的水平,是野毒菌株的89%。CD1小鼠毒力试验结果显示,srtA基因缺失株SC211的LD50(4.93×107)约为野毒菌株SC21的LD50(8.21×106)的6倍,质粒介导的互补菌株SC212的LD50(1.43×107)是野生菌株SC21的1.7倍,接近野毒菌株的水平,说明srtA基因缺失后SS2毒力显著下降。

In the present study, srtA gene mutant strain was constructed by using the thermo-sensitive suicide vector pSET4s and confirmed by PCR and Southern blotting. The results showed that the construction of the mutant strain was successful, named SC211. In addition complementation mutant strain was constructed by using the E. coli/ Streptococcus shuttle vector pAT18, named SC212. The characteristic of SC211 and SC212 was further researched. The virulence of different strains was determined by animal infection,which demonstrated that the virulence was decreased clearly when the srtA gene knocked out. The virulence of the mutant was restored when strain mediated by plasmid-containing the srtA gene. However, it could not reach the level of the wild type. The hemolytic activity of the srtA gene mutant strain and the complementation strain had no difference when compared with the wild type strain. The adherence to the Hep2 cell of the srtA gene mutant strain degraded obviously,and the adherence to the Hep2 cell of the complementation strain had no obvious difference compared with the wild type.