奶牛乳房炎金黄色葡萄球菌荚膜多糖蛋白结合疫苗的研究

发布时间：2018-03-28 23:13

本文选题：奶牛乳房炎　切入点：金黄色葡萄球菌　出处：《甘肃农业大学》2017年硕士论文

【摘要】：本研究以奶牛乳房炎金黄色葡萄球菌临床分离株J581为研究对象,应用单因素及正交优化试验,对四种培养基进行了筛选,培养条件进行了优化。利用透射电镜,对优化前后条件下培养的细菌荚膜情况进行了比较观察。培养基筛选结果表明,哥伦比亚液体培养基最适合金黄色葡萄球菌的生长,与培养条件优化前相比,菌体产量提高了20%,且增加了有荚膜菌体的数量和荚膜厚度。对通过细菌发酵液吸光度预估菌液活菌数方法进行了研究,开展了J581株对小鼠致病性的研究。试验成功构建了通过菌液吸光度预估活菌数方法,得出金黄色葡萄球菌对小鼠的半数致死量为4.83×108CFU。发酵液经离心后,采用高压裂解法从菌体沉淀中提取多糖,并利用苯酚抽提法去除蛋白。结果,从6L发酵液中获得含28.15%的多糖共1.534 g,经苯酚抽提后,蛋白质含量为3.44%。将提取的荚膜多糖粗提物,采用琼聚糖凝胶柱进行了荚膜多糖纯化,纯化后的多糖经溴化氰活化后与己二酰肼(ADH)形成多糖-酰肼基衍生物,在碳二亚胺(EDAC)作用下与破伤风类毒素共价进行结合。利用紫外全波长扫描,对多糖蛋白偶联情况进行了鉴定,同时将偶联物冻干,注射用盐水溶解制得金黄色葡萄球菌多糖蛋白结合疫苗。结果表明:经琼聚糖凝胶CL-4B纯化冻干后,测得荚膜多糖的终产量为34.79 mg/L,蛋白含量为0.74%,核酸含量为0.92%,唾液酸含量为12.29%。与多糖最大吸收峰相比,偶联物的吸收峰发生了偏移,表明多糖蛋白偶联成功。制备出了荚膜多糖蛋白结合疫苗。本项研究建立了金黄色葡萄球菌最佳的培养条件和荚膜多糖提取、纯化工艺流程,制备出了奶牛乳房炎金黄色葡萄球菌荚膜多糖蛋白结合疫苗,为今后进一步深入开展乳房炎疫苗研究,很好的预防奶牛乳房炎具有重要的意义。
[Abstract]:In this study, the clinical isolate J581 of Staphylococcus aureus in dairy cow mastitis was used as the research object. Four kinds of culture medium were screened by single factor and orthogonal optimization test, and the culture conditions were optimized by transmission electron microscope (TEM). The culture conditions of bacteria capsule before and after optimization were compared and observed. The results of medium screening showed that the Colombian liquid medium was the most suitable for the growth of Staphylococcus aureus, and compared with that before the optimization of culture conditions. The cell yield was increased by 20%, and the number and thickness of the capsule were increased. The method of estimating the number of viable bacteria by the absorbance of bacterial fermentation broth was studied. The pathogenicity of strain J581 to mice was studied. The method of estimating the number of live bacteria by absorbance of bacteria solution was successfully constructed. The median lethal dose of Staphylococcus aureus to mice was 4.83 脳 10 8 CFU. After centrifugation, the fermentation broth was centrifuged. Polysaccharide was extracted from bacterial precipitate by high pressure pyrolysis and protein was removed by phenol extraction. Results the polysaccharide containing 28.15% was obtained from 6L fermentation broth, and the protein content was 3.44% after phenol extraction. The crude extract of capsule polysaccharide was extracted from 6L fermentation broth. The perfringent polysaccharides were purified by agarose gel column. The purified polysaccharides were activated with cyanide bromide to form polysaccharide acyl hydrazide derivatives. The conjugation of carbodiimide (EDAC) with tetanus toxoid was carried out. The coupling of polysaccharides and proteins was identified by UV full-wavelength scanning, and the coupling compound was freeze-dried. The polysaccharide protein binding vaccine of Staphylococcus aureus was prepared by dissolution of saline solution for injection. The results showed that the vaccine was freeze-dried after purification by CL-4B. The final yield of capsule polysaccharide was 34.79 mg / L, the protein content was 0.74, the nucleic acid content was 0.92and the sialic acid content was 12.29.The absorption peak of the conjugate was shifted compared with the maximum absorption peak of polysaccharide. The results showed that the conjugation of polysaccharides and proteins was successful. The best culture conditions of Staphylococcus aureus and the extraction and purification process of capsule polysaccharides were established. The preparation of bovine mastitis Staphylococcus aureus capsule polysaccharide protein binding vaccine is of great significance for the further research of mastitis vaccine and the prevention of cow mastitis.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

【相似文献】