透明质酸生产菌溶血素S基因缺失突变菌株的构建及其特性

Construction and characterization of hemolysin S gene mutant strain producing hyaluronic acid

摘要:

[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sagA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pJR700载体系统，构建马链球菌兽疫亚种sagA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sagA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sagA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sagA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sagA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sagA不仅是表达溶血素SLS的基因，同时sagA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。

Abstract:

[Objective] Streptococcus equi subsp. zooepidemicus (GCS) is mainly used to produce hyaluronic acid (HA) in the industry. GCS secretes the hemolysis toxin (streptolysin S, SLS) that causes hemolysis in the host cells. Therefore, the safety of HA produced by GCS is concerned. We constructed an engineering strain, to produce commercial HA without SLS by knocking out sagA.[Method] The sagA of GCS was knocked out by the thermosensitive delivery vector system pJR700. The sagA mutant was identified through PCR with primers homologous to the flanking regions and SLS analysis. The yield of HA, HA molecular weight and virulence factors such as streptolysin Hylc, hyaluronate lyase, glyceraldehyde-3-phosphate dehydrogenase and cell surface proteins were determined by spectrophotometer and SDS-PAGE.[Result] We constructed successfully the in-frame deletion sagA mutant strain of GCS. In the sagA mutant, HA titer increased more than 30% than that of the wild type strain and no SLS hemolytic activity was detected. Compared to the wild type strain the sagA mutant decreased the quality of surface proteins, hemolytic Hylc activity and glyceraldehyde-3-phosphate dehydrogenase activity. The activities of hyaluronidase and cell were increased in the sagA mutant.[Conclusion] The sagA not only expressed hemolysis S but also regulated production of HA, the quality of surface proteins and activities of hyaluronidase, hemolysis Hylc and glyceraldehydes-3-phosphate dehydrogenase in Streptococcus equi subsp. zooepidemicus.