【作者】 史冬艳；

【导师】 郝永清；

【作者基本信息】 内蒙古农业大学 ， 预防兽医学， 2010， 博士

【摘要】 奶牛乳房炎不仅给世界乳品工业带来巨大的经济损失,而且还危害到公共卫生和人类健康。其中金黄色葡萄球菌是引起奶牛乳房炎的主要病原,由于金黄色葡萄球菌具有在乳腺细胞内定殖的能力以及对抗生素极易产生耐药性的特性,使得用抗生素治疗的效果越来越差,因此使用疫苗进行防治就成为首选。研究表明,引起奶牛乳房炎的金黄色葡萄球菌的耐药性随地域的不同而发生显著变化。为此,本研究从内蒙古呼和浩特地区患有乳房炎的奶牛采取样本,利用常规微生物学方法,分离到60株金黄色葡萄球菌。利用K-B琼脂法,对20种抗生素进行了耐药性分析,分别为:青霉素G、氨苄西林、哌拉西林、阿莫西林、头孢氨苄、头孢噻肟、头孢他啶、头孢唑林、头孢西丁、复方新诺明、卡那霉素、庆大霉素、环丙沙星、诺氟沙星、氧氟沙星、呋喃唑酮(痢特灵)、罗红霉素、克林霉素、红霉素、万古霉素。实验结果显示,90%以上的金黄色葡萄球菌分离株耐青霉素G,即使是对G+菌有较强抑杀作用的林可胺类和大环内酯类,也出现了高达50%左右的耐药率。此外,金黄色葡萄球菌对复方新诺明(60%),呋喃唑酮(痢特灵)(60%),罗红霉素(51.67%)和氨苄西林(51.67%)也出现了较高的耐药率。大量研究证实,金黄色葡萄球菌的黏附素识别并特异性的结合到组织以及细胞的特定分子上是感染的关键,因此阻断细菌的黏附和在粘膜表面的定殖,可能是预防感染最有效的策略。凝集因子A(ClfA)、纤连蛋白A(FnBPA)和纤连蛋白B(FnBPB)是和金黄色葡萄球菌致病性相关的重要黏附因子。凝集因子A的A区(ClfA-A)和纤维蛋白原(Fg)结合,纤连蛋白A的D区(FnBPA-D)和纤连蛋白B的D区(FnBPB-D)主要和纤维结合素(Fn)结合。本研究通过PCR扩增技术,对临床分离到的60株金黄色葡萄球菌凝集因子A的A区和纤连蛋白D区进行了特异性的扩增,结果表明所有分离株全部含有凝集因子A的基因,并且59株分离株至少含有纤连蛋白A和B中的一种,80%的菌株含有编码FnBPA-D和FnBPB-D的基因。虽然上述三种黏附素对金黄色葡萄球菌性乳房炎的致病作用在国内外已有报道,但有关这三种黏附素进行混合免疫的研究,目前的国内外研究尚无报道。因此,本实验利用pET-32a表达载体成功表达了金黄色葡萄球菌ClfA-A(990bp) ,FnBPA和FnBBP的D片段(402bp),并通过免疫家兔对重组融合蛋白pET32-ClfA、pET32-FnBPA和pET32-FnBPB的特异性抗体产生的能力、单一抗血清抗黏附能力,以及不同黏附素抗体组合的抗黏附能力和吞噬调理水平进行了系统研究。实验结果显示,所有经重组蛋白免疫的家兔均可以产生较高水平的特异性抗体,而且抗体均显示出较强的抑制金黄色葡萄球菌黏附到纤维蛋白原和纤维结合素的作用。而且联合免疫组(三种重组蛋白进行混合免疫)的抗黏附效果优于其它单一抗原免疫组。此外,调理吞噬实验结果显示,免疫兔全血对金黄色葡萄球菌有较强的调理作用,且混合免疫组的兔全血(含三种不同黏附素的抗体的混合物)的吞噬调理作用最强。本研究在国内率先应用ClfA-A、FnBPA-D和FnBPB-D基因构建亚单位疫苗,并系统地开展了实验动物免疫研究,探讨了上述三种黏附素的不同免疫组合的免疫效果。本研究将为奶牛乳房炎金黄色葡萄球菌基因工程疫苗的研制提供新的思路。更多还原

【Abstract】 Mastitis is one of the largest production concerns in the dairy industry worldwide. Staphylococcus aureus (S.aureus) is the most common etiologic agent of mastitis in bovines and a major cause of economic losses in the dairy industry. The occurrence of bovine mastitis has serious consequences for public health. The effects of antibiotic therapy has becoming more and more worse because S.aureus has the capacity of adherence and anchorage to mammary gland epithelium and the Characteristic of easy to produce antibiotic-resistant strains. Thus, a vaccine to protect against infection would therefore be beneficial. Antimicrobial susceptibility of S.aureus isolated from bovine mastitis varies widely by region. Therefore, 60 strains S.aureus were isolated from cases of mastitis in cow herds of Inner Mongolia of China by using microbiological method. Susceptibility to ampicillin, penicillin G, amoxicillin, piperacillin , cephalexin, cephazolin, cefotaxime, ceftazidime, cefoxitin, SMZ-TMP, gentamycin, kanamycin, norfloxacin, ciprofloxacin, ofloxacin, furaxone, torlamician, roxithromycin ,clindamycin, vancomycin, determined by the K-B agar method. Antibiotic susceptibility testing showed over 90% isolates were resistant to penicillin G. Also, high level of resistance to SMZ-TMP(57.20%), furaxone(58.47%), roxithromycin(58.90%), vancomycin(53.81%) and ampicillin(47.88%) was observed among S.aureus strains.In this study, Fn-binding region D of FnBPs and fibrinogen-binding region A of ClfA were detected by PCR from clinical strains isolated from China. The results showed 59 tested strains carried at least one fnb gene and all of 60 strains tested carried region A of clfa.80% isolated strains carried FnBPA-D and FnBPB-D.A large number of research shows that recognizing and specifically anchoring of S.aureus adhesins to tissues and definite molecule of cell is one of the key steps in the initiation of an infection. Blocking the primary stages of S.aureus infection, specifically the bacterial adhesin to cell and the colonization of the mucosal surface, may be the most effective strategy for preventing infections. Clumping factor A (ClfA), fibronectin binding protein A (FnBPA) and fibronectin binding protein B(FnBPB) of S.aureus are important proteins involved in the pathogenesis of staphylococcal bovine mastitis which promote binding of fibrinogen and fibrin to the bacterial cell surface. ClfA contains region A that binds fibrinogen and is a target for vaccine development. Fibronectin binding proteins FnBPA and FnBPB contains the Fn-binding domain D which promote binding of fibrin.Numerous studies have underlined the role of ClfA, FnBPA and FnBPB in the staphylococcal infectious process, but there isn’t the report on immunization effect of different immunization strategy. Thus, the region A of ClfA (ClfA-A) and region D of FnBPA(FnBPA-D) and FnBPB(FnBPB-D) of S.aureus were successfully expressed in expression vector pET-32a and detect the anti adhesiveness, specific antibody and opsonophagocytic of antiserum of the recombinant fusion protein in the present study.The results showed that sera from recombinant adhesin-immunized rabbit were more efficient than sera from PBS immunized rabbit at inhibiting bacterial adherence and producing of specific antibody (p < 0.01). Study proved the mixture of three kinds of recomimant proteins has an advantage in assey of antiadhesion effects. Moreover, opsonophagocytic test result showed that whole blood of immuned rabbit possessed effect of eliminating S.aureus, and opsonization of the mixture of three kinds of vaccinated rabbit blood was significantly higher than the other group immuned with single antigen.It is the first time to application ClfA-A, FnBPA-Dand FnBPB-D genes of S.aureus to construct subunit vaccine,and the systematic immune animal experiments were carried out to examine the effection of different immunization strategy in this paper.Thus, the study will provide new breakthrough ideas for cow mastitis S.aureus genetic engineering vaccine research.更多还原