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
发布时间：2019-02-13 20:35
【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。
[Abstract]:In order to investigate the effects of cpsE and neuA on the biological characteristics of Streptococcus niloticus (GBS) capsule polysaccharides synthesis genes, homologous recombination was used to construct the single gene deletion mutant of cpsE and neuA of GBS. The specific methods are as follows: construction of cpsE and neuA gene knockout recombinant plasmids pSET4s-cpsE and pSET4s-neuA. with chloramphenicol resistance gene by Infusion-PCR The constructed plasmids were electrically transformed into GBS receptive cells, and the double exchange and plasmid loss were realized by changing the culture temperature. Finally, the suspected knockout strain was obtained by chloramphenicol resistance screening. The suspected knockout strain was verified by colony PCR,RT-PCR and DNA sequencing. The results showed that two GBS mutants 螖 cpsE and 螖 neuA were successfully constructed. On the basis of biological function analysis, the difference of growth rate, capsule polysaccharide thickness, sialic acid content and virulence between the mutant 螖 cpsE, 螖 neuA and wild strain was compared. The results showed that the growth rate of 螖 cpsE and 螖 neuA was not significantly different from that of wild strain, but the thickness of capsule polysaccharide, the content of sialic acid and virulence of strain were significantly lower than that of wild strain. Further studies showed that cpsE was a key gene for the synthesis of GBS capsule polysaccharides from fish, while neuA gene was a key gene for sialic acid acidification of capsule polysaccharides. Their deletion resulted in a decrease in the content of sialic acid in the capsule of GBS and significantly reduced the virulence of the strain.
【作者单位】： 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;上海海洋大学水产与生命学院;
【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)
【分类号】：S943
[Abstract]:In order to investigate the effects of cpsE and neuA on the biological characteristics of Streptococcus niloticus (GBS) capsule polysaccharides synthesis genes, homologous recombination was used to construct the single gene deletion mutant of cpsE and neuA of GBS. The specific methods are as follows: construction of cpsE and neuA gene knockout recombinant plasmids pSET4s-cpsE and pSET4s-neuA. with chloramphenicol resistance gene by Infusion-PCR The constructed plasmids were electrically transformed into GBS receptive cells, and the double exchange and plasmid loss were realized by changing the culture temperature. Finally, the suspected knockout strain was obtained by chloramphenicol resistance screening. The suspected knockout strain was verified by colony PCR,RT-PCR and DNA sequencing. The results showed that two GBS mutants 螖 cpsE and 螖 neuA were successfully constructed. On the basis of biological function analysis, the difference of growth rate, capsule polysaccharide thickness, sialic acid content and virulence between the mutant 螖 cpsE, 螖 neuA and wild strain was compared. The results showed that the growth rate of 螖 cpsE and 螖 neuA was not significantly different from that of wild strain, but the thickness of capsule polysaccharide, the content of sialic acid and virulence of strain were significantly lower than that of wild strain. Further studies showed that cpsE was a key gene for the synthesis of GBS capsule polysaccharides from fish, while neuA gene was a key gene for sialic acid acidification of capsule polysaccharides. Their deletion resulted in a decrease in the content of sialic acid in the capsule of GBS and significantly reduced the virulence of the strain.
【作者单位】： 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;上海海洋大学水产与生命学院;
【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)
【分类号】：S943
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