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【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】 The purpose of this study was to clone the SspA truncated gene of Streptococcus suis serotype 2 Streptococcus suis serotype 2 to verify whether it has the enzymatic activity and to construct the bacterium of the deletion mutants of this gene. The role of the disease process [. Methods The prokaryotic expression plasmid of SspA truncated gene 05SSU0811 of SS2 was constructed and expressed and purified. The serine protease substrate Succinyl-Ala-Pro-Phe-p-nitroanilide (pNa) was used to detect the expression product The homology recombination technique was used to knock out 05SSU0811 gene. The knockout strain was screened by multiple crossover PCR and sequenced. The animal experiment was conducted to analyze the influence of 05SSU0811 gene deletion on bacterial virulence. 【Result】 05SSU0811 protein was successfully expressed and purified at a concentration of about 3.5 g / L. Serine protease activity test confirmed that it has enzymatic activity; obtained 05SSU0811 gene deletion mutant, the mice challenge test showed that all 20 wild-type mice were dead, gene-deficient mutant infected group died 9 Rate of 45%, there was a significant difference between the two groups in mortality. The results showed that the virulence of the strain with the deletion of 05SSU0811 gene was significantly lower than that of the wild-type strain. 【Conclusion】 The truncated serine protease encoded by 05SSU0811 gene still has enzymatic activity. The truncated SspA gene of SS2 has a certain role in the pathogenicity of the highly pathogenic Streptococcus suis type 2 S. suis.