目的 丝/苏氨酸激酶(STK)与丝/苏氨酸磷酸酶(STP)通过对蛋白质的可逆磷酸化调控原核生物的各项生理活动.文中旨在研究2型猪链球菌中国强毒株05ZYH33中、stk/stp1同时缺失对其生物学特性及致病性的影响.方法 运用同源重组的方法构建2型猪链球菌中国强毒株05ZYH33的stk/stp1基因敲除突变株 Δstk/stp1,比较分析野毒株05ZYH33与突变株 Δstk/stp1的生物学特性;通过细胞黏附实验、抗吞噬实验以及小鼠感染模型分析stk/stp1缺失对细菌毒力的影响.将30只SPF级BALB/c小鼠,用随机数字表法分成05ZYH33组(注射1 mL野毒株菌液)、Δstk/stp1组(注射1 mL突变株菌液)和THB组(注射1 mL的新鲜THB液体培养基),每组10只.结果 PCR结果显示,stk/stp1基因被壮观霉素抗性基因Spcr替换,突变株构建成功;生物学特性实验表明,05ZYH33和 Δstk/stp1在接种2 h后开始缓慢增殖,在7 h时都到达平台期.突变株进入对数生长期(A600≈0.4)的时间比野毒株推迟1 h左右,到达平台期后的菌体密度比野毒株低(0.8 vs 1.0).05ZYH33和 Δstk/stp1在血平板上均长出灰白色、圆形半透明、湿润、表面光滑的细小菌落.菌落周围有明显的 β-溶血环,菌落形态和溶血活性无明显变化.小鼠致病性实验显示,05ZYH33组小鼠在12 h内全部死亡,Δstk/stp1组小鼠在12 h内死亡9只,24 h内全部死亡,THB组无发病和死亡症状.结论 stk/stp1同时缺失可能主要影响细菌增殖、分裂相关蛋白的调控.
Objective Serine /threonine kinases (STK) and phosphatases (STP) regulate various physiological activities of prokaryotes by reversible phosphorylation of proteins .This paper aimed to study the effects of simultaneous deletion of the stk and stp1 genes on the biological characteristics and pathogenicity of streptococcus suis type 2, the Chinese virulent strain 05ZYH33. Methods The double mutant of the stk and stp1 genes of 05ZYH33 was constructed by homologous recombination .The biological characteristics of the wild strain 05ZYH33 and the mutant strain Δstk/stp1 were compared.The effects of the stk and stp1 deletion on bacterial virulence was analyzed using cell adhesion assay , anti-phagocytosis assay and the mouse model of infection . Results RT-PCR showed that the stk and stp1 genes were replaced by the spectinomycin resistance gene Spc r and the mutant strain was successfully constructed .Experi-ments of biological characterization revealed gradually increased value of 05ZYH33 and Δstk/stp1 at 2 hours after inoculation and a plateau period at 7 hours.The logarithmic phase of the mutant strain (A600≈0.4) was 1 hour later than that of the wild one , and the bacterial den-sity of the former was lower than that of the latter after the plateau pe -riod (0.8 vs 1.0).On the blood plates of 05ZYH33 and Δstk/stp1 were observed greyish, round, semitransparent, wet and smooth-sur-faced tiny bacterial colonies , around which there were hemolysis rings with no significant differences in colony morphology and hemolytic ac -tivity.In the experiment on pathogenicity , the mice of the 05ZYH33 group all died within 12 hours while 9 of the 30 mice in the Δstk/stp1 group died within 12 hours and all died within 24 hours. Conclusion The simultaneous deletion of the stk and stp1 genes may mainly affect the regulation of the proteins associated with bacte -rial proliferation and division.