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【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】 To construct Vir D4 knockout mutant of type IV secretion system component of serotype 2 virulent strain 05ZYH33 of Streptococcus suis serotype 05, preliminary analysis of its activity and virulence, in order to further study Streptococcus suis type 2 To evade the role of the host natural immune killer provide the basis. 【Method】 Using the genome of 05ZYH33 as a template, the upstream and downstream homologous arms of Vir D4 gene were amplified by PCR. The shuttle plasmid p SET1 was used as a template to amplify the chloramphenicol resistance gene Cm. The three fragments were constructed by overlap PCR Was ligated to the thermosensitive vector p SET4s to construct the gene knockout vector p SET4s :: Vir D4; and the gene knockout mutant ΔVir D4 was constructed by homologous recombination; the in vitro whole blood killing experiment, the CD1 mouse competition infection and the challenge experiment were performed on the mutation The virulence of strains and wild strains were compared. 【Result】 The gene knockout mutant ΔVir D4 was obtained. The virulence of the mutant was lower than that of the wild-type strain. 【Conclusion】 Vir D4, a type Ⅳ secretion system of Streptococcus suis, is related to its virulence and plays a role in early resistance to natural immune cell killing.