2型猪链球菌Fbps基因敲除突变体的构建及毒力分析

Construction and Virulence Evaluation of Gene Fbps Knock-Out Mutant of Streptococcus suis Type 2

目的:敲除2型猪链球菌(SS2)强毒株05ZYH33中的Fbps基因,研究该基因敲除对菌株生物活性及毒力的影响,为深入探讨猪链球菌致病机制提供实验基础。方法:从05ZYH33基因组中扩增Fbps基因上、下游同源臂,从pSET1质粒中扩增氯霉素抗性基因Cm,通过重叠PCR的方法将3个片段整合后连接到温敏自杀载体pSET4s上,电转入05ZYH33感受态细胞,通过改变培养温度实现双交换和质粒丢失,最后经抗性筛选获得敲除株05ZYH33ΔFBPS,分析敲除株的生物学性状,以CD1小鼠作为体外感染模型对突变株和野生株进行毒力比较。结果:PCR分析和测序结果均显示Fbps基因敲除成功,动物实验结果显示Fbps基因敲除后05ZYH33的毒力有所下降。结论:与野生株相比,突变株对小鼠的毒力有所降低。

Objective： Knocking out gene Fbps from Streptococcus suis 2（SS2） strain 05ZYH33 and evaluating its biological activity in order to provide an experimental basis for studying the pathogenesis of SS2.Methods： Us ing genomic DNA of 05ZYH33 as template to amplify the up and down fragments of Fbps,pSET1 plasmid as tem plate were used to amplify the chromosomal（Cm） resistance cassette.By overlap extension PCR method,recombi nant gene knock-out vector was constructed consisting of Cm cassette with flanking homology regions to the target gene.The plasmid pSET4s-FBPS was transformed into wild-type strain 05ZYH33,by changing temperature the double-crossover was achieved and plasmid loss,the gene knock-out mutant strain 05ZYH33ΔFBPS was screened.Biological characteristics and virulence of mutant strain in mice were compared with wild-type strain 05ZYH33.Results Conclusion： PCR analysis and sequencing confirmed that the gene Fbps was replaced completely by Cm cassette.The biological characteristics and virulence of the mutant strain 05ZYH33ΔFBPS was lower than the wild-type strain 05ZYH33.