唾液乳杆菌素SJH-8的分离纯化及其生物学特性与氨基酸序列研究

发布时间：2018-02-26 17:17

  本文关键词： 抗生素 唾液乳杆菌SJH-8 细菌素 生物学特性 纯化 氨基酸序列　出处：《武汉纺织大学》2017年硕士论文　论文类型：学位论文

【摘要】：抗生素的使用,为人类、动植物预防及治疗病菌感染等疾病做出了重要贡献,但由于抗生素难降解,使环境遭到严重破坏与污染,且已诱发多种抗性基因的产生,人类的身体健康受到严重的威胁。乳酸菌素作为一种多肽或蛋白类物质,可被酶水解,是一种环境友好型抗菌物质,具有替代抗生素使用的潜力。我国乳酸菌资源丰富,研究稳定、高效、广谱、无抗性的乳酸菌素近年来成为热点。研究多以分离纯化、生物学特性、作用机理为主,而对其氨基酸序列及基因序列等信息获得较少,难以对其进行量化,从而为其应用带来一定的困难。因此,本课题着手于开发一种新型的乳酸菌素,并研究其氨基酸序列及基因序列,为今后的异源表达构建重组工程菌株来生产细菌素提供有力的前提条件。本课题首先从珠江口水产动物肠道中分离乳酸菌,以副溶血弧菌(Vibro parahaemolyticus ATCC 17802)和金黄色葡萄球菌(Staphylococcus aureus ATCC35844)为指示菌,以筛选具有广谱抑菌活性的菌株。然后对该乳酸菌所产细菌素的生物学特性进行研究,并分离纯化该细菌素,后通过SDS-PAGE及MALDI-TOF/TOF质谱检测推测出细菌素的氨基酸序列,并以特异引物扩增出表达该细菌素的基因,为细菌素替代抗生素的应用提供依据,并为其生产提供前提条件。具体研究结果如下:1.从珠江口水产动物健康罗非鱼、白斑鱼、南美白对虾肠道中共筛选出97株乳酸菌,以V.parahaemolyticus和S.aureus为指示菌、用滤纸片琼脂扩散法做抑菌活性初筛和复筛后,获得4株对两种指示菌均有抑菌活性的乳酸菌,其中菌株SJH-8抑菌活性最好,因此选用该菌作为研究细菌素的供试菌。基于菌株的16S r DNA序列构建系统发育树,确定SJH-8为一株唾液乳杆菌。2.经硫酸铵盐析后对阴性菌有抑制作用的活性部分仍处于上清中,而沉淀样品对多种革兰氏阳性致病菌有较强的抑菌活性,对阴性菌无抑菌活性。沉淀样品中的活性成分可被胰蛋白酶完全水解,经木瓜蛋白酶、胃蛋白酶、蛋白酶K处理后活性有所减弱;有较强的热稳定和p H稳定性,于121℃下处理15 min或p H2.0和p H10.0处理2 h,仍保留较好活性。3.经SDS-PAGE和MALDI-TOF/TOF质谱检测,推测出了细菌素SJH-8的部分氨基酸序列,并确定该细菌素是由SJH-8α和SJH-8β两条多肽组成的ClassⅡb类肽。后根据基因abp118设计特异引物,以基因SJH-8的DNA为模板进行PCR扩增,最后得到表达细菌素的基因序列。
[Abstract]:The use of antibiotics has made important contributions to the prevention and treatment of human, animal and plant diseases such as bacterial infections. However, due to the difficult degradation of antibiotics, the environment has been seriously damaged and polluted, and many resistance genes have been induced. As a kind of polypeptide or protein substance, lactic acid bacteriocin can be hydrolyzed by enzyme, it is a kind of environment-friendly antimicrobial substance and has the potential to replace antibiotics. The study of stable, efficient, broad-spectrum, non-resistant lactic acid bacillins has become a hot topic in recent years. Most of the studies focus on isolation and purification, biological characteristics, action mechanism, but less information on amino acid sequence and gene sequence. It is difficult to quantify it, which brings some difficulties to its application. Therefore, this paper begins to develop a new type of lactic acid bacteriocin, and study its amino acid sequence and gene sequence. In order to construct the recombinant engineering strain to produce bacteriocin in the future, we first isolated lactic acid bacteria from the aquatic animal intestine of the Pearl River Estuary. Vibro-Vibro parahaemolyticus ATCC 17802) and Staphylococcus aureus ATCC35844) were used to screen the strains with broad-spectrum antibacterial activity. The biological characteristics of bacteriocin produced by the Lactobacillus were studied, and the bacteriocin was isolated and purified. The amino acid sequence of bacteriocin was detected by SDS-PAGE and MALDI-TOF/TOF mass spectrometry, and the gene expressing bacteriocin was amplified with specific primers, which provided the basis for the application of bacteriocin in the substitution of antibiotics. The specific results are as follows: 1.A total of 97 strains of lactic acid bacteria were screened from the intestines of healthy tilapia, white spotted fish and Penaeus vannamei in the Pearl River Estuary, with V. parahaemolyticus and S. aureus as indicators. Four lactic acid bacteria with antimicrobial activity were obtained by using filter paper Agar diffusion method, and four strains of lactic acid bacteria were obtained after screening and rescreening. Among them, the strain SJH-8 had the best bacteriostatic activity. The phylogenetic tree was constructed based on the 16s r DNA sequence of the strain, and SJH-8 was identified as a Lactobacillus saliva strain. The inhibitory activity of SJH-8 on negative bacteria was still in the supernatant after ammonium sulfate salting out. However, the precipitated samples have strong bacteriostatic activity against various gram-positive pathogens, but no inhibitory activity against negative bacteria. The active components in the precipitated samples can be completely hydrolyzed by trypsin, and can be hydrolyzed by papain, pepsin, pepsin, pepsin, papain, pepsin, papain, pepsin, pepsin. The activity of protease K was weakened after treatment with protease K, and had strong thermal stability and pH stability. After being treated at 121 鈩，

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