尼罗罗非鱼无乳链球菌基因缺失株△cpsE和△neuA的构建及其生物学特性

摘要

为探究尼罗罗非鱼无乳链球菌(GBS)荚膜多糖合成基因cpsE和neuA对菌株生物学特性的影响,本研究利用同源重组的方法,构建了GBS的cpsE与neuA的单基因缺失突变株.具体方法为:用Infusion-PCR的方法分别构建带有氯霉素抗性基因的cpsE与neuA基因敲除重组质粒pSET4s-cpsE和pSET4s-neuA.将构建好的质粒电转化入GBS感受态细胞中,通过改变培养温度实现双交换和质粒丢失,最后经氯霉素抗性筛选获得疑似敲除株.通过菌落PCR、RT-PCR及DNA测序等方法对疑似敲除株进行验证.结果显示GBS的两个突变株△cpsE和△neuA被成功构建.在此基础上,通过生物学功能分析比较基因缺失突变株△cpsE、△neuA与野生株在菌株生长速率、荚膜多糖厚度、唾液酸含量和毒力方面的差异.结果发现缺失突变株△cpsE和△neuA的生长速度与野生株无显著差异,但荚膜多糖厚度、唾液酸含量和菌株毒力均显著低于野生株.进一步研究显示,cpsE是鱼源GBS荚膜多糖合成的关键基因,neuA基因则是荚膜多糖唾液酸化的关键基因,它们的缺失导致了GBS荚膜唾液酸含量的降低,且显著降低了菌株的毒力.%To investigate the functions of the capsular polysaccharide synthetic genes cpsE and neuA of Streptococcus agalactiae (GBS) isolated from Nile tilapia (Oreochromis niloticus),two single-gene knockout mutant strains,namely △cpsE and △neuA,were constructed by homologous recombination.Genomic DNA of GBS was used as a template to amplify the up and down homologous fragments of cpsE and neuA,whereas the pSET1 plasmid was used as a template to amplify the chromosomal chloramphenicol resistance gene (cat).Two recombinant gene knockout plasmids,pSET4s-cpsE and pSET4s-neuA,both containing cat,were constructed by the In-Fusion(R) polymerase chain reaction (PCR) method.The recombinant plasmids pSET4s-cpsE and pSET4s-neuA were transformed into wild-type GBS by electroporation.Double-crossover and plasmid loss strains were obtained by changing the culture temperature.Finally,△cpsE and △neuA were screened for chloramphenicol resistance and the mutations were confirmed by PCR,real-time PCR,and DNA sequencing.To characterize △cpsE and △neuA,their growth rate,capsule thickness,capsular sialic acid content,and virulence were compared with those of wild-type GBS.The results showed that the growth rates of the wild-type,△cpsE,and △neuA strains did not significantly differ.However,the capsule thickness,capsular sialic acid content,and virulence of △cpsE and △neuA were significantly lower than those of the wild-type strain.Further research suggested that cpsE is the critical synthetic gene of the capsular polysaccharide of GBS,whereas neuA is important for capsular polysaccharide sialylation.The deletion of cpsE and neuA not only significantly reduced the capsular sialic acid content of GBS isolated from fish,but also significantly impaired its virulence.