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【目的】马链球菌兽疫亚种是工业上生产透明质酸的主要菌种,该菌能产生引起宿主细胞溶血的链球菌溶血素S(streptolysin S,SLS)毒素,因而其产品的安全性一直是人们所担心的问题。本实验的目的就是通过基因敲除的方法构建不产SLS的透明质酸生产工程菌,同时探讨溶血素sag A基因缺失对菌株透明质酸合成和其他毒力因子的影响。【方法】利用温度敏感/自杀性质粒p JR700载体系统,构建马链球菌兽疫亚种sag A基因缺失突变株;通过PCR扩增,溶血平板和SLS含量测定等方法确定sag A基因缺失;采用分光光度、SDS-PAGE和细胞毒性试验等分析方法,对野生菌株和sag A基因缺失突变菌株透明质酸含量、透明质酸分子量、溶血素Hylc、透明质酸分解酶、甘油醛-3-磷酸脱氢酶和菌体表面蛋白等相关毒力因子进行对比研究。【结果】获得了透明质酸产量提高30%而溶血活性极低的马链球菌兽疫亚种sag A基因缺失突变株。该突变株与野生菌株相比较,透明质酸分解酶活性增加而透明质酸相对分子量降低,此外,与毒力相关的表面蛋白含量、溶血素Hylc和甘油醛-3-磷酸脱氢酶活性也显著降低。细胞毒性实验结果表明,野生菌株与sag A基因缺失突变菌株的培养物上清液,对细胞活性的影响存在显著差异。【结论】在马链球菌兽疫亚种中sag A不仅是表达溶血素SLS的基因,同时sag A基因对菌株透明质酸合成、透明质酸分解酶、菌体表面蛋白、溶血素Hylc和甘油醛-3-磷酸脱氢酶等都具有调节作用。
【Aims】 Streptococcus equi subsp. Zooepidemicum is a major strain of hyaluronic acid produced industrially. The strain can produce streptolysin S (SLS) toxin, which causes hemolysis of host cells. Therefore, the safety of its products has been People are worried about the problem. The objective of this experiment was to construct a non-SLS-producing hyaluronan-producing bacterium by gene knockout method and to explore the effect of sag A gene deletion on the synthesis of hyaluronan and other virulence factors. 【Method】 The sag A gene deletion mutant of Streptococcus equi subsp. Zebrai was constructed by using temperature-sensitive / suicide plasmid p JR700 vector system. The deletion of sag A gene was determined by PCR amplification, determination of hemolytic plate and SLS content. The hyaluronic acid content, hyaluronic acid molecular weight, hemolysin Hylc, hyaluronolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase Hydrogenase and cell surface proteins and other related virulence factors were compared. 【Result】 The sag A gene deletion mutant of Streptococcus equi subsp. Zebrae was obtained with a 30% increase in the production of hyaluronic acid and a very low hemolytic activity. Compared with the wild strain, the mutant strain increased the activity of hyaluronolytic enzyme and decreased the relative molecular weight of hyaluronic acid. In addition, the surface protein content related to virulence, hemolysin Hylc and glyceraldehyde-3-phosphate dehydrogenase activity Significantly lower. Cytotoxicity test results showed that there was a significant difference in the effects on the cell viability between the wild strain and the sag A gene deletion mutant culture supernatant. 【Conclusion】 sag A is not only the gene for hemolysin SLS expression in the zebrafish subsp. Faecium subsp. Equisimilis, but also sag A gene is highly sensitive to the strain hyaluronan synthesis, hyaluronolytic enzyme, cell surface protein, hemolysin Hylc and glyceraldehyde -3-phosphate dehydrogenase all have a regulatory role.