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

发布时间：2018-08-05 18:25

【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。
[Abstract]:[objective] Streptococcus equi is the main strain producing hyaluronic acid in industry. It can produce streptococcal hemolysin (S (streptolysin) toxin which causes hemolysis of host cells. The aim of this study was to construct engineering bacteria that did not produce SLS with hyaluronic acid by gene knockout. At the same time, the effects of hemolysin sag A gene deletion on hyaluronic acid synthesis and other virulence factors were investigated. [methods] using the temperature sensitive / suicide plasmid p JR700 vector system, the mutant of sag A gene deletion of Streptococcus equi subspecies was constructed. The deletion of sag A gene was determined by PCR amplification, hemolysis plate and SLS assay, and the hyaluronic acid content of wild strain and mutant strain of sag A gene was determined by spectrophotometry SDS-PAGE and cytotoxicity test. Hyaluronic acid molecular weight, hemolysin Hylc, hyaluronidase, The virulence factors such as glyceraldehyde-3-phosphate dehydrogenase and bacterial surface protein were compared. [results] the mutant of sag A gene of Streptococcus equistii subspecies was obtained, which increased hyaluronic acid production by 30% and had very low hemolytic activity. Compared with wild strain, the activity of hyaluronidase increased and the relative molecular weight of hyaluronic acid decreased. In addition, the virulence related surface protein content, hemolysin Hylc and glyceraldehyde 3-phosphate dehydrogenase activity also decreased significantly. The results of cytotoxicity test showed that the culture supernatants of wild strain and sag A gene deletion mutant strain were supernatant. [conclusion] sag A is not only the gene that expresses hemolysin SLS, but also sag A gene on hyaluronic acid synthesis, hyaluronidase and cell surface protein in Streptococcus equi. Hemolysin Hylc and glyceraldehyde-3-phosphate dehydrogenase have regulatory effects.
【作者单位】：西南交通大学生命科学与工程学院;
【基金】：国家自然科学基金(31070117) 四川省科技支撑项目(2012FZ0048)~~
【分类号】：TQ281;Q93

【相似文献】