猪链球菌2型Ⅳ型分泌系统组分VirD4与毒力相关性分析

Correlation between Type Ⅳ secretion system component VirD4 and virulence for Streptococcus suis 2

【目的】构建猪链球菌2型强毒株05ZYH33毒力岛89 K上的Ⅳ型分泌系统组分Vir D4敲除突变株,初步分析其活性和毒力,为进一步研究猪链球菌2型在逃避宿主天然免疫杀伤中的作用提供基础。【方法】以05ZYH33基因组为模板,PCR扩增Vir D4基因上下游同源臂,以穿梭质粒p SET1为模板,PCR扩增氯霉素抗性基因Cm,通过重叠PCR技术搭建上述3个片段并连接至温敏载体p SET4s,构建基因敲除载体p SET4s∷Vir D4;通过同源重组构建基因敲除突变株ΔVir D4;通过体外全血杀伤实验、CD1小鼠竞争感染及攻毒实验对突变株和野生株的毒力进行比较分析。【结果】获得了基因敲除突变株ΔVir D4,通过对比发现其毒力与野生株相比有所降低。【结论】猪链球菌2型Ⅳ型分泌系统组分Vir D4与其毒力相关,并在早期抵抗天然免疫细胞杀伤中发挥一定作用。

[ Objective] In order to study the role of SS2 Type 1V Secretion System VirD4 in evasion of the host innate immune killing, we constructed a knockout mutant ΔVirD4. Then we studied its biological activity and virulence. [Methods] The two VirD4 flanking DNA sequences were amplified using genome of 05ZYH33 as template. We also amplified the Cm sequence of shuttle vector pSET1, and through overlap extension PCR we connected the three fragments together. Using suicide vector pSET4s, we constructed the recombinant gene knockout vector pSET4s ：： VirD4. The mutant ΔVirD4 was successfully constructed by allelie replacement. Virulence of mutant strain was compared with wild type strain 05ZYH33 through in vitro bactericidal assays, competitive infection and challenge experiment of CD1 mice. [ Results] Mutant strain AVirD4 was constructed successfully, its virulence attenuated compared to the wild type strain. [ Conclusion] These findings indicated that Type IV Secretion System component VirD4 contributed to the virulence of S. suis with important functions in evading innate immunocyte killing.