發(fā)布時間：2018-03-28 23:13

  本文選題：奶牛乳房炎　切入點：金黃色葡萄球菌　出處：《甘肅農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：本研究以奶牛乳房炎金黃色葡萄球菌臨床分離株J581為研究對象,應(yīng)用單因素及正交優(yōu)化試驗,對四種培養(yǎng)基進行了篩選,培養(yǎng)條件進行了優(yōu)化。利用透射電鏡,對優(yōu)化前后條件下培養(yǎng)的細(xì)菌莢膜情況進行了比較觀察。培養(yǎng)基篩選結(jié)果表明,哥倫比亞液體培養(yǎng)基最適合金黃色葡萄球菌的生長,與培養(yǎng)條件優(yōu)化前相比,菌體產(chǎn)量提高了20%,且增加了有莢膜菌體的數(shù)量和莢膜厚度。對通過細(xì)菌發(fā)酵液吸光度預(yù)估菌液活菌數(shù)方法進行了研究,開展了J581株對小鼠致病性的研究。試驗成功構(gòu)建了通過菌液吸光度預(yù)估活菌數(shù)方法,得出金黃色葡萄球菌對小鼠的半數(shù)致死量為4.83×108CFU。發(fā)酵液經(jīng)離心后,采用高壓裂解法從菌體沉淀中提取多糖,并利用苯酚抽提法去除蛋白。結(jié)果,從6L發(fā)酵液中獲得含28.15%的多糖共1.534 g,經(jīng)苯酚抽提后,蛋白質(zhì)含量為3.44%。將提取的莢膜多糖粗提物,采用瓊聚糖凝膠柱進行了莢膜多糖純化,純化后的多糖經(jīng)溴化氰活化后與己二酰肼(ADH)形成多糖-酰肼基衍生物,在碳二亞胺(EDAC)作用下與破傷風(fēng)類毒素共價進行結(jié)合。利用紫外全波長掃描,對多糖蛋白偶聯(lián)情況進行了鑒定,同時將偶聯(lián)物凍干,注射用鹽水溶解制得金黃色葡萄球菌多糖蛋白結(jié)合疫苗。結(jié)果表明:經(jīng)瓊聚糖凝膠CL-4B純化凍干后,測得莢膜多糖的終產(chǎn)量為34.79 mg/L,蛋白含量為0.74%,核酸含量為0.92%,唾液酸含量為12.29%。與多糖最大吸收峰相比,偶聯(lián)物的吸收峰發(fā)生了偏移,表明多糖蛋白偶聯(lián)成功。制備出了莢膜多糖蛋白結(jié)合疫苗。本項研究建立了金黃色葡萄球菌最佳的培養(yǎng)條件和莢膜多糖提取、純化工藝流程,制備出了奶牛乳房炎金黃色葡萄球菌莢膜多糖蛋白結(jié)合疫苗,為今后進一步深入開展乳房炎疫苗研究,很好的預(yù)防奶牛乳房炎具有重要的意義。
[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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.23

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