假交替单胞菌胞外产物的特性分析及其抑菌蛋白的克隆与表达

发布时间：2018-04-10 12:37

本文选题：假交替单胞菌 + 胞外产物　；参考：《上海海洋大学》2017年硕士论文

【摘要】：第一部分益生菌在水产养殖中的应用水产养殖业迅速发展,病害频发,利用益生菌来改善生态环境、预防治疗水产动物疾病已经越来越得到认可。本部分对水产益生菌的定义、水产养殖中常用益生菌种类以及益生菌的作用机制简单概述,并介绍了假交替单胞菌属,该属能够分泌多种生物活性物质,包括小分子化合物、蛋白质、多糖等,在水产病害防治、赤潮防治等方面具有较大的潜力。第二部分假交替单胞菌m8和a22胞外产物和胞内产物抑菌活性比较本部分运用玻璃纸覆盖平板法制备实验菌株的胞外产物和胞内产物,通过滤纸片法检测胞外产物和胞内产物的抑菌活性,结果表明胞外产物和胞内产物对副溶血弧菌都有抑制作用,且胞外产物的抑菌效果要好于胞内产物。为了优化制备胞外产物的培养时间,分别于培养12h、24h、36h和48h后制备胞外产物,然后用滤纸片法进行抑菌活性检查,并对各个时间制备的胞外产物总蛋白含量进行定量。结果表明,培养24h后制备胞外产物的抑菌活性最好且总蛋白含量在24h也达到最大值。第三部分假交替单胞菌m8和a22胞外产物相关性质研究将过氧化氢酶浓度设为三个梯度,分别为100μg/mL,200μg/mL,500μg/mL,测试了过氧化氢酶对胞外产物抑菌活性的影响。结果显示过氧化氢酶能够抑制胞外产物的抑菌活性,含有过氧化氢酶的滤纸片能够使抑菌圈产生缺口,但可能是滤纸片距离摆放的原因,不同浓度的过氧化氢酶产生缺口并没有明显的差异。L-氨基酸氧化酶能够催化氨基酸释放过氧化氢而产生抑菌作用,但它们的底物多种多样。用修改后的分光光度计法检测过氧化氢的产生量,将20种L-氨基酸分别作为底物,在505nm下测吸光值。结果显示,L-赖氨酸作为底物产生过氧化氢的量高于其他L-氨基酸,菌株m8的胞外蛋白添加L-赖氨酸产生过氧化氢量高于a22,这与m8的抑菌活性好于a22相对应,推测通过产生过氧化氢来产生抑菌活性。通过变性聚丙烯酰氨凝胶电泳(SDS-PAGE)及胶内活性检测,发现分子量为70KDa的蛋白条带具有抑菌活性,在胶条上形成明显的抑菌条带,并将该条带切下来进行质谱分析,结果表明,菌株m8的70KDa蛋白条带鉴定为TonB-dependent receptor[Pseudoalteromonas flavipulchra JG1],菌株a22的70KDa蛋白条带鉴定为抗菌蛋白antibacterial protein[Pseudoalteromonas flavipulchra JG1]。第四部分菌株a22抑菌蛋白的克隆与表达根据Gene Bank公布的抑菌蛋白的序列,用PrimerPremier5.0软件设计引物,以菌株基因组DNA为模板扩增目的基因,将纯化后的目的基因与表达载体p BAD/gⅢA连接,转入大肠杆菌BL21,构建重组表达菌株BL21/p BAD/gⅢA/2-p-1,在20℃用终浓度为0.5%L-阿拉伯糖进行诱导表达,表达产物经过SDS-PAGE显示该蛋白能在大肠杆菌中异源表达,但是表达产物的可溶部分并未检测到抑菌活性,推测可能是形成了包涵体,无法进行翻译后修饰,导致表达产物没有抑菌活性。
[Abstract]:The first part of the application of probiotics in aquaculture aquaculture development, frequent occurrence of diseases, to improve the ecological environment of the use of probiotics, prevention and treatment of aquatic animal diseases has been increasingly recognized. This part of the definition of aquatic bacteria and probiotics, mechanism of probiotics briefly commonly used in aquaculture health benefits, and introduces false alternate Aeromonas genus, the genus can secrete a variety of bioactive substances, including small molecules, proteins, polysaccharides, disease prevention and control in aquaculture, has great potential in the aspect of red tide prevention. The second part Pseudoalteromonas M8 and A22 extracellular products and the intracellular antibacterial activity of this part of the application of glass paper cover plate prepared by the experimental strain of extracellular products and intracellular product, antibacterial activity by filter paper method for the detection of extracellular products and intracellular products. The results show that extracellular products And intracellular product on Vibrio parahaemolyticus has better antibacterial effect and inhibition of extracellular products from intracellular products. In order to develop the time to optimize the preparation of extracellular products, were cultured 12h, 24h, preparation of extracellular products of 36h and 48h, and then check the antibacterial activity by filter paper method. The total protein content of extracellular products and the preparation time quantitatively. The results showed that after 24h culture, preparation of extracellular products the best antibacterial activity and total protein content in 24h can reach the maximum. The third part Pseudoalteromonas M8 and A22 extracellular products on some properties of catalase concentration for the three gradient, respectively 100 g/mL, 200 g/mL, 500 g/mL, the effect of hydrogen peroxide on extracellular enzyme product antibacterial activity were tested. The results showed that catalase can inhibit the antibacterial activity of extracellular products, filter paper containing catalase can make the inhibition Bacteria produce ring gap, but may cause the filter paper from the display, the catalase concentration had different gap and no difference of.L- amino acid oxidase was capable of catalyzing amino acid release of hydrogen peroxide produced inhibitory effect, but their substrates varied. With modified spectrophotometric method for determination of hydrogen peroxide production, will 20 kinds of amino acids were L- as substrate, the absorbance measured at 505nm. The results showed that L- lysine as substrate to produce hydrogen peroxide was higher than that of other L- amino acids, strain M8 protein added L- lysine producing hydrogen peroxide was higher than that of A22, and the antibacterial activity of M8 is better than A22 corresponding it suggests that the production of hydrogen peroxide to produce antibacterial activity. By denaturing polyacrylamide gel electrophoresis (SDS-PAGE) detection and in gel activity, found that the molecular weight of 70KDa protein bands with antibacterial activity, in Glue to form a significant inhibition zone, and the strip cut down by mass spectrometry analysis, the results show that the strain M8 70KDa protein band was identified as TonB-dependent receptor[Pseudoalteromonas flavipulchra JG1], A22 strain 70KDa protein band was identified as antibacterial protein[Pseudoalteromonas flavipulchra JG1]. cloning antibacterial protein fourth strain A22 inhibitory protein and expression of the the sequence of Gene inhibitory protein released by Bank, primers were designed by PrimerPremier5.0 software according to the genomic DNA as template to amplify the target gene, the gene expression vector and purified P BAD/g III A connection into E.coli BL21. The recombinant expression strains of BL21/p BAD/g III A/2-p-1 at 20 DEG C, with a final concentration of 0.5%L- in Arabia sugar induced expression product by SDS-PAGE showed that the protein was heterologously expressed in Escherichia coli, However, the soluble part of the expressed product did not detect antibacterial activity. It was presumed that the inclusion body was formed and could not be modified after translation, resulting in no antibacterial activity of the expression product.

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S948;S963.211

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