2型猪链球菌gene0969基因敲除突变体的构建及毒力分析

Construction and Virulence Analysis of gene0969 Gene Knock-Out Mutant of Streptococcus suis Serotype 2

目的:构建2型猪链球菌强毒株05ZYH33毒力岛89K上的Ⅳ型分泌系统组分gene0969敲除突变体,初步分析其活性,为进一步研究猪链球菌假想毒力因子在致病中的作用提供实验基础。方法:以05ZYH33基因组为模板,PCR扩增gene0969基因的上下游片段;以穿梭质粒pSET1为模板,PCR扩增氯霉素抗性基因Cm;采用重叠PCR方法搭建3个片段,并克隆到自杀载体pSET4s上,构建基因敲除载体,通过同源重组构建gene0969突变体,再用小鼠感染模型对突变株和野生株的毒力进行比较。结果:获得了gene0969基因敲除突变体,并发现其毒力与野生型相比有下降趋势。结论:2型猪链球菌假想毒力因子gene0969可能与毒力有关,其作用和机制值得进一步分析。

Objective：To construct gene knock-out mutant of a type Ⅳ secretion system component gene0969 which is located on candidate pathogenicity island 89K of Streptococcus suis serotype 2（SS2） virulent strain 05ZYH33 and evaluate its biological activity in order to provide an experimental basis for studying the role of putative virulence factors in SS2 pathogenesis.Methods： The flanking DNA sequence of gene0969 was amplified from the chromosomal DNA of S.suis 05ZYH33,the Cm gene cassette amplified from the E.coli-S.suis shuttle vector pSET1,through overlap extension PCR method,recombinant gene knock-out vector was constructed consisting of Cm cassette with flanking homology regions to the target gene,and isogenic gene0969-deficient mutant was screened by allelic replacement.The comparison of the mutant and wild type was analysed by the mice infection model.Results： The knock-out mutant of gene0969 was constructed successfully and its virulence attenuated than wild type.Conclusion： The putative virulence factor gene0969 contributes to the virulence of SS2,and the role and mechanism of this protein is valuable for further analysis.