高致病性2型猪链球菌截短型类枯草杆菌丝氨酸蛋白酶基因的鉴定和功能研究

Identification and functional study of the truncated surface-associated subtilisin-like protease gene of Streptococcus suis serotype 2

【目的】克隆表达高致病性2型猪链球菌05ZYH33株的SspA截短型基因,验证其是否具有酶学活性,并构建该基因的缺失突变株细菌,探讨其在2型猪链球菌致病过程中所起的作用【。方法】构建SS2的SspA截短型基因05SSU0811原核表达质粒,表达并纯化05SSU0811蛋白,运用丝氨酸蛋白酶底物Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide(pNa),通过显色反应检测表达产物的酶学活性;运用同源重组技术敲除05SSU0811基因,多重交叉PCR筛选敲除株并测序鉴定,动物试验分析05SSU0811基因缺失对细菌毒力的影响。【结果】成功表达并纯化05SSU0811蛋白,浓度约为3.5 g/L。丝氨酸蛋白酶活性测定试验证实其具有酶学活性;获得05SSU0811基因缺失突变株,小鼠攻毒试验表明,野生株攻毒的20只小鼠全部死亡,基因缺失突变株攻毒组死亡9只,死亡率45%,两组间死亡率有显著性差异。表明05SSU0811基因缺失的菌株毒力较野生株明显下降。【结论】05SSU0811基因编码的截短型丝氨酸蛋白酶仍然具有酶学活性,SS2的截短型基因SspA在高致病性2型猪链球菌的致病性方面具有一定作用。

[Objective] To clone and express the truncated surface-associated subtilisin-like protease gene 05SSU0811 of Streptococcus suis serotype 2 （SS2） highly virulent strain 05ZYH33 and measure the activity of the recombinant protease; Construct the 05SSU0811 gene knockout mutant strain and analyze its contribution to pathogenicity. [Methods] The 05SSU0811 gene encoding SspA was amplified and cloned into the expression plasmid pET28a and then transformed into Escherichia coli BL21 to overproduce the protein. The recombinant protease was purified by chromatography procedures. Its activity was measured by using the chromogenic substrate Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide （pNa）. The 05SSU0811 gene was replaced with spectinomycin resistance cassette through homologous recombination, then multiple-PCR and sequence analysis were adopted to identify the knockout strain ？05SSU0811. The virulence of SS2 wild type and ？05SSU0811 mutant strain were then evaluated by calculating the survival rate of the infected mice. [Results] The recombinant SspA was expressed and purified. Its activity was demonstrated by the subtilisin-like protease assay. The isogenic mutant ？05SSU0811 was successfully constructed and the virulence of the ？05SSU0811 mutant strain was attenuated remarkably compared to the wild type strain. [Conclusion] The truncated SspA encoded by 05SSU0811 gene in SS2 exhibits its activity in vitro. And it also contributes to the virulence of SS2.