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

摘要:

【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。

Abstract:

[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 plcR gene, a foreign gene exclusively existed in virulent SS2 strains, plays an important role in the pathogenesis of SS2 infection.