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研究

发布时间：2018-03-08 09:04

本文选题：透明质酸　切入点：溶血素-S　出处：《西南交通大学》2016年硕士论文　论文类型：学位论文

【摘要】：存在于生物机体各个组织器官中的透明质酸是一种酸性黏多糖。由于该物质具有优良粘弹性和高度水合性的理化性质,从而能越来越广泛的应用于临床医学、研究医学、化妆品及食品药品添加剂等相关领域,因此,在科学研究上具有极大价值。在工业生产上,马链球菌兽疫亚种是发酵生产透明质酸的重要菌种之一。但由于该菌有一定的致病性,长期以来,人们担心利用该菌株发酵生产得到透明质酸产品的安全能否有保证。特别是该菌株所产生毒力因子——溶血素-S(SLS)能够造成典型的细胞溶血和细胞膜损坏。因此,我们要求发酵法获得的透明质酸中不能含有链球菌SLS毒素。研究发现,在化脓性链球菌中溶血素-S的基因sagA是一调控基因,能够调控透明质酸及毒力因子的表达。我们推测在sagA基因结构相似的马链球菌兽疫亚种中,sagA基因同样能够调控透明质酸及毒力因子的表达。本论文通过采用温度敏感自杀载体系统和电转化的方法构建了马链球菌兽疫亚种sagA基因缺失突变菌株和其回复突变菌株,使得HA生产菌不产溶血素-S。同时利用不同的方法对马链球菌兽疫亚种相关毒力因子进行了研究。结果发现,相对于野生菌来说,sagA基因缺失突变菌中HA产量提高了30%、分子量下降了一个数量级。另外,与毒力相关的甘油醛-3-磷酸脱氢酶、Hylc、菌体表面蛋白含量显著降低,透明质酸分解酶含量增加。通过细胞实验发现,菌体的总毒力明显下降。利用单因素实验和正交实验进一步使透明质酸产量提高了20%左右。本研究证明,在马链球菌兽疫亚种中sagA基因不仅表达溶血素-S,同时对毒力因子具有调控作用。sagA基因的缺失,对菌株透明质酸和透明质酸分解酶具有上调作用,对甘油醛-3-磷酸脱氢酶、Hylc、菌体表面蛋白等毒力因子具有下调作用。
[Abstract]:Hyaluronic acid, which exists in various tissues and organs of organism, is an acidic mucopolysaccharide. Due to its excellent viscoelastic and highly hydrated physicochemical properties, hyaluronic acid can be more and more widely used in clinical medicine and medical research. Cosmetics, food and drug additives and other related fields, therefore, in scientific research has great value. In industrial production, Streptococcus equi is one of the most important strains of hyaluronic acid in fermentation. However, because of its pathogenicity, Streptococcus equi has long been responsible for the production of hyaluronic acid. There is concern that the safety of producing hyaluronic acid products by using the strain can be guaranteed. In particular, the virulence factor produced by the strain-hemolysin-SLSs-can cause typical cell hemolysis and cell membrane damage. We require that hyaluronic acid obtained by fermentation do not contain streptococcus SLS toxin. Studies have found that the gene sagA of hemolysin S is a regulatory gene in Streptococcus pyogenes. The expression of hyaluronic acid and virulence factor can be regulated. We speculate that SagA gene can also regulate the expression of hyaluronic acid and virulence factor in the subspecies of Streptococcus equi with similar structure of sagA gene. In this paper, we use temperature to control the expression of hyaluronic acid and virulence factor. The mutant strain of sagA gene deletion and its reverse-mutation strain of Streptococcus equi subspecies were constructed by using the sensitive suicide vector system and electrical transformation method. At the same time, different methods were used to study the virulence factors related to the subspecies of Streptococcus equi. Compared with wild bacteria, HA production increased by 30% and molecular weight decreased by one order of magnitude. In addition, the virulence related to glyceraldehyde 3 phosphate dehydrogenase Hylc, the protein content on cell surface decreased significantly. The content of hyaluronidase was increased. The total virulence of the cell was obviously decreased by cell experiment. The yield of hyaluronic acid was further increased by 20% or so by single factor experiment and orthogonal experiment. In the subspecies of Streptococcus equi, sagA gene not only expressed hemolysin -S, but also regulated the deletion of virulence factor. It also up-regulated hyaluronic acid and hyaluronidase. It can down-regulate virulence factors such as glyceraldehyde-3-phosphate dehydrogenase Hylc and cell surface protein.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q93

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