尼罗罗非鱼无乳链球菌基因缺失株ΔcpsE和ΔneuA的构建及其生物学特性  

Construction and characterization of cpsE-and neuA-deleted mutants of Streptococcus agalactiae isolated from Nile tilapia

作　　者：师红亚 董浚键[1] 张德锋[1] 孙成飞[1] 田园园[1] 曾庆凯[1] 卢迈新[1] 叶星[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所农业部热带亚热带水产资源利用与养殖重点实验室,广东广州510380 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《中国水产科学》2017年第5期977-987,共11页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(NSFC)(31272688);现代农业产业技术体系建设专项资金项目(CARS-48);中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金项目(2017YH-ZC06)

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ΔcpsE和ΔneuA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ΔcpsE、ΔneuA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ΔcpsE和ΔneuA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 Strepto- coccus agalactiae （GBS） isolated from Nile tilapia （Oreochromis niloticus）, two single-gene knockout mutant strains, namely AcpsE and AneuA, 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 recombi- nant gene knockout plasmids, pSET4s-cpsE and pSET4s-neuA, both containing cat, were constructed by the In-Fusion 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, AcpsE and AneuA were screened for chloramphenicol resistance and the mutations were confirmed bY PCR, real-time PCR, and DNA sequencing. To characterize AcpsE and AneuA, 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, AcpsE, and AneuA strains did not sig- nificantly differ. However, the capsule thickness, capsular sialic acid content, and virulence of AcpsE and AneuA 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 sia- lylation. The deletion of cpsE and neuA not only significantly reduced the capsular sialic acid content of GBS iso- lated from fish, but also significantly impaired its virulence.

关 键 词：尼罗罗非鱼 无乳链球菌 荚膜多糖合成基因 基因敲除 生物学特性 

分 类 号：S917[农业科学—水产科学]