高致病性2型猪链球菌plcR基因敲除株的构建及其相关生物学特性的研究

Construction and characterization of plcR gene knockout mutant of Streptococcus suis serotype 2

【目的】构建高致病性2型猪链球菌05ZYH33菌株plcR基因敲除株,通过比较突变株与野生株生物学特性的差异,研究plcR基因在2型猪链球菌致病过程中的作用。【方法】利用同源重组技术敲除plcR基因,多重交叉PCR及RT-PCR鉴定并测序验证。比较野生株与突变株基本生物学特性的差异,小鼠攻毒实验分析plcR基因缺失对细菌毒力的影响。【结果】经RT-PCR证实05SSU0241与05SSU0242共转录,通过多重交叉PCR及RT-PCR证实成功构建plcR基因缺失突变株,基本生物学特性显示突变株的生长速率、菌落形态、溶血活性均无显著改变,小鼠致病性试验结果显示,野生株攻毒的小鼠死亡率为70%,突变株攻毒的小鼠死亡率为40%,毒力较野生株显著降低。【结论】plcR基因作为2型猪链球菌有毒株基因组中特有的外源基因,在细菌致病过程中具有重要作用。

[Objective] To construct the plcR gene knockout mutant of Streptococcus suis serotype 2 （SS2） highly virulent strain 05ZYH33 and analyze the role of plcR in the pathogenesis of SS2. [Methods] The plcR gene was replaced with a spectinomycin resistance cassette through homologous recombination, then multiple-PCR, RT-PCR, and sequence analysis were performed to identify the knockout strain △plcR. The effects of plcR deletion on the basic biological characteristics and virulence of SS2 were then determined in this study. [Results] The results of RT-PCR confirmed that 05SSU0241 and 05SSU0242 should be transcribed as a single operon. The isogenic mutant △plcR was successfully constructed verified by multiple-PCR and RT-PCR. No significant differences in biological characteristics, including growth rate, colony morphology and hemolytic activity, were detected between the two strains. Pathogenic trial in mouse showed that wild-type strain induced high fatality rate as much as 70%, whereas the △plcR mutant caused a 40% fatality rate. Obviously that the virulence of the △plcR mutant was attenuated remarkably compared with the wild type. [Conclusion] The pleR gene, a foreign gene exclusively existed in virulent SS2 strains, plays an important role in the pathogenesis of SS2 infection.