2型猪链球菌SSU0448基因敲除突变株的构建及其毒力分析

Construction and virulence analysis of the SSU0448 gene knockout mutant in Streptococcus suis serotype 2

摘要:

【目的】 通过构建2型猪链球菌(SS2)强毒株05ZYH33的SSU0448基因缺失突变株Δ0448和互补株CΔ0448，探索SSU0448基因缺失对细菌基本生物学特性和细菌毒力的影响。【方法】用同源重组基因敲除方法构建筛选强毒株05ZYH33中N-乙酰半乳糖胺和半乳糖胺代谢途径相关转录调节因子SSU0448基因的缺失突变株，比较分析突变株Δ0448与野生株05ZYH33、互补株CΔ0448的基本生物学特性，小鼠毒力实验分析SSU0448基因缺失对细菌毒力的影响。【结果】PCR检测分析显示，SSU0448基因在转化重组体中被壮观霉素抗性基因所替代，表明基因敲除突变株构建成功；同时构建了基因功能互补株CΔ0448。生物学特性实验表明突变株Δ0448在成链能力上较野生株明显减弱，对数生长期稍短，快速到达平台期；而菌落形态、革兰氏染色和溶血活性方面无明显差异；小鼠毒力实验发现，突变株毒力并无显著改变。【结论】SSU0448基因的敲除能够改变2型猪链球菌的成链能力；不影响其侵袭致病能力，可能延缓2型猪链球菌的发病过程，此研究为2型猪链球菌致病感染奠定了基础。

Abstract:

[Objective] Gene knockout mutant Δ0448 and the functional complementary strain CΔ0448 were constructed to investigate the role of SSU0448 gene on Streptococcus suis serotype 2 (SS2) morphology and virulence. [Methods] Based on the principle of homologous recombination, the SSU0448 gene was knocked out in SS2 virulent strain 05ZYH33. PCR analysis was carried out to identify the mutant, then screened the functional complementary strain CΔ0448 containing the SSU0448 gene expressing plasmid. The morphology and virulence of the established strains were examined. [Results] PCR analysis and DNA sequencing identified the knockout mutant Δ0448 and the SSU0448 gene was completely replaced by the spcr cassette. The functional complementary strain CΔ0448 was also successfully constructed. Gram staining showed that the mutant Δ0448 grew much shorter than the wild type 05ZYH33 and faster to reach the plateau phase. Deletion of the SSU0448 gene did not change the virulence of SS2 in mice infection assays. [Conclusion] Knockout of SSU0448 gene obviously influenced the chain ability of Streptococcus suis serotype 2, and its deficiency has no significant effect on the virulence in the mice model while it may delay the pathogenesis.