2型猪链球菌SSU05_0736基因敲除株的构建及其特性分析

Construction and characterization of c AMP receptor protein knock-out mutant of Streptococcus suis serotype 2

目的 2型猪链球菌是重要的人畜共患病病原菌,鉴定现有的和发掘新的毒力因子依然是该领域的研究热点。探讨SSU05_0736编码的c AMP受体蛋白在2型猪链球菌(Streptococcus suis serotype 2,S.suis 2)中国强毒株05ZYH33中的作用机制,初步分析SSU05_0736基因敲除株的生物学特性,为进一步研究猪链球菌可能的毒力因子及其致病机制提供研究基础。方法利用同源重组原理构建并筛选由壮观霉素抗性基因(Spcr)替换S.suis 2中SSU05_0736的基因敲除株Δ0736,系统比较分析野毒株与敲除株的生物学特性,同时将BALB/c小鼠作为动物感染模型来探究敲除株的毒力。结果通过组合PCR、反转录PCR(RT-PCR)等方法鉴定并成功构建基因敲除株Δ0736,其在菌落形态、溶血活性及对小鼠致病力等方面与野毒株无显著差异;两者生长速率相比,敲除株的生长较野毒株迟缓但其在对数期增长速率较大。结论 SSU05_0736基因的缺失并未使猪链球菌2型强毒株05ZYH33的毒力发生显著改变,推测SSU05_0736基因并非毒力决定因子,但与野毒株相比敲除株在生长速率尤其对数期增长速率较大这一方面的改变,提示可对其代谢调控能力作进一步分析。

Objective Streptococcus suis serotype 2 （S. suis2） is an important zoonotic pathogen. The identification of the existing and new virulence factors remains a hot topic in the field. This study aimed to investigate the action mechanisms of the Crp/Fnr family transcriptional regulator cAMP receptor protein （cAMP receptor protein, Crp） encoded by SSU05_0736 in S. suis2, the Chinese virulent strain 05ZYH33, and analyze the virulence and biological features of the mutant strain so as to provide an experimental basis for further studies of S. suis2 hypothetical virulence factors and their pathogenesis. Methods Using the homologous recombination principle, we recombined the knock-ont mutant A0736, substituted its encoding gene for Spcr Cassette, and then compared the biological characteristics and the virulence in the murine model between the mutant A0736 and the wild-type strain 05ZYH33. Results Multiple PCR and reverse transcription PCR （RT-PCR）confirmed that the SSU05_0736 gene was completely replaced by the Spc＇ cassette, showing the successful construction of the knock-out mutant strain A0736. No significant differences were observed between A0736 and 05ZYH33 in terms of colony morphology, hemolytic activity, and pathogenicity in mice. However, the former grew more slowly but had a higher growth rate in the log phase than the latter. Conehlslon The deletion of the SSU05_0736 gene does not change the virulence of S. suis2 05ZYH33 significantly, indicating that SSU05_0736 is not the decisive factor of the virulence. The difference in the growth rate, especially in the log phase, suggests that further analysis can be performed on the metabolic regulation capacity of the knock-out mutant strain.