【作者】 杨明锋；

【导师】 陈创夫；

【作者基本信息】 石河子大学 ， 预防兽医学， 2010， 硕士

【摘要】 奶牛乳房炎是影响奶牛养殖业最重要的疾病之一,可以引起产奶量及乳品质量下降,花费巨额的治疗费用而给奶牛养殖业带来巨大的经济损失。奶牛乳房炎是多病因疾病,因为乳房炎发生过程中有大量的微生物参与,使用疫苗预防也就成为乳房炎防控的一个重要策略。金葡菌是引起奶牛乳房炎与乳品微生物污染的主要病原菌之一,发展有效的金葡菌疫苗是控制临床型乳房炎的关键,已经有疫苗得到了推广应用。随着生物技术的发展,新型疫苗不断涌现,科研人员也用新技术去研究金葡菌疫苗。随着对金葡菌侵染过程的了解,人们认识到金葡菌黏附到细胞或细胞外基质上是感染的关键步骤,用特异性抗体阻断金葡菌的黏附过程可能为金葡菌乳房炎的防治提供一个有效的策略,参与细菌黏附过程的分子就成为潜在的抗原分子。已知参与金葡菌黏附的主要分子有纤维结合素结合蛋白(FnBP)、凝集因子A (ClfA)、凝集因子B(ClfB)、胶原连接蛋白(Can)等。本研究采用原核表达的FnBP和ClfA蛋白,纯化后制备亚单位疫苗,免疫小鼠进行疫苗评价。因为金葡菌是典型的胞外寄生菌,体液免疫对预防金葡菌感染起重要作用,IL-6的作用之一就是促进抗体的产生,本研究克隆表达出奶牛白细胞介素6(IL-6)作为亚单位疫苗的佐剂。用小鼠进行乳房炎疫苗效果评价,要先建立小鼠乳房炎模型,取泌乳8-10天的小鼠,第四对乳腺内注射不同剂量的金葡菌或PBS,2h后母子合笼,24h后处死母鼠,取乳腺进行一侧甲醛固定进行病理检测,另外一侧研磨检测组织中金葡菌的数量和TNF-α的浓度。用纯化的FnBP和C1fA蛋白加入氟氏不完全佐剂制备亚单位疫苗,培养金葡菌收菌后通过反复冻融超声破碎加入氟氏不完全佐剂制备全菌疫苗,间隔14天免疫小鼠两次,一部分使怀孕,泌乳后攻菌,另一部分加强免疫时注射不同的佐剂(PHA,LPS,IL-6,IFN-γ,胸腺肽),免疫不同时间点采血分离血清,用ELISA法检测血清中抗体水平。结果成功克隆表达了奶牛IL-6基因,并纯化出蛋白；建立了金葡菌感染小鼠乳房炎模型,确定1.0×103cfu／只金葡菌是合适的乳腺内感染剂量,和评价小鼠乳房炎的严重程度三个有效指标。免疫结果显示,亚单位疫苗和全菌疫苗都可以减轻乳腺内感染金葡菌造成的乳房炎严重程度,表现为病理变化轻,组织中金葡菌数量少,TNF-α水平低。以全菌总蛋白为抗原,用琼扩试验检测血清中的抗体,只有全菌免疫组有可见的沉淀线,以纯化的两种蛋白包板,用ELISA检测血清中的抗体显示亚单位疫苗组的抗体水平最高。而5种不同种类的佐剂对血清中抗体的水平有不同的影响,其中以胸腺肽增加血清中抗体的作用最明显。更多还原

【Abstract】 Bovine mastitis, one of the most significant disease of dairy herds, has huge effects on farm economics due to reduction in milk production and treatment costs. It’s a multiple reasons disease, thus we must Application of a variety of measures to control bovine mastitis.As there are many kinds of Microorganism have a role in the processes of mastitis, the vaccine become a important target for control bovine mastitis.Staphylococcus aureus (S. aureus) is the main causative agent of bovine mastitis, development an efficient vaccine of S.aureus is important for control the infection,there are several vaccines used in commercially herds, but the economic benefit following such treatment has not been uniformly demonstrated. With the development of biotechnology, new type vaccine emerged one after another, researchers also used these new technology for the vaccine ’invention. Followed the knowledge improve about the S.aureus infected in mammary gland, peoples found it’s the key step of S.aureus adhesion to the host celles or extracelluar matrix. The adhesion molecules yet become the potential candidate antigens. As is know about the molecules include the Fibronectin binding protein (FnBP), clumping factor A (ClfA),clumping factor B(ClfB), Collagen-binding protein Can (Can) et al.In this study, we choose prokaryotic express proteins of FnBP and ClfA, purificated for prepared the subunit vaccine. Evaluate the effectiveness on mouse model. S. aureus is a bacteria planted out of the host celles, Humoral immune palys an important role in resistancing S. aureus infection. As well know it have an important role to enhance the IgG level. That we clones the IL-6 gene of cows and expressed in prokaryotic express system. Using the IL-6 as an adjuvant for subunit vaccine. We established a mastitis mouse model before to evaluated the vaccine, take mice at 8-10days during lactating, injection different dose S. aureus or PBS into the fourth pair mammary glands,2 hours later take mother-children co-cage,24 hours apart after injection, killed mice by cervical pull, remove one side of mammary gland fixed for histopathologic evaluations, another side pulverization to detect the bacteria amount and the tumor necrosis factor (TNF-α) concentration. Through the mouse model we gained the suitable dose of S. aureus to challenge after vaccination. Take purificated proteins add adjuvant prepared subunit vaccine, and collection S. aureus lysis using Ultrasonic, add adjuvant to prepared total bacteria vaccine. Mice were vaccinated twice 14 days apart, one group cohabit to pregnancy, after parturition give S. aureus injection to challenge evaluation, another group give different adjuvant (PHA, LPS, IL-6, IFN, thymosin) with second vaccinated, haemospasia and apart serum every week, detect the IgG level in serum used the ELISA.Results, we got purificated bovine IL-6, through clone and prokaryotic expression, established a mastitis mouse model, gained the suitable dose of S. aureus was 1.0×103cfu per mouse.and the scientific criteria to evaluated the severity of mouse mastitis. Both of the two vaccine can significantly reduce the severity of mastitis in mouse model, with a light histopathologic changes, lower TNF-αlevel and a small amount of bacteria in mammary gland tissues.The total protein vaccine group shown a highest total IgG level, but the subunit vaccine group shown a highest anti-FnBP & ClfA antibody, every adjuvant can enhance the IgG level rising significantly, the thymosin was the best one between the five adjuvants.更多还原