奶牛乳房炎病原基因ClfA、LPS和hlb真核表达体系的建立及ClfA免疫保护效果初步评价

发布时间：2018-03-10 13:30

本文选题：奶牛乳房炎　切入点：病原　出处：《甘肃农业大学》2016年博士论文　论文类型：学位论文

【摘要】：奶牛乳房炎是造成奶牛饲养行业经济损失的重要原因之一。经过多年的认识和研究,已经建立起来了比较完备的预防和控制体系;但是,在畜牧业相对落后的发展中国家,由于疾病防控理论和实践相对落后,该病造成的经济损失依然非常严重。减少和杜绝奶牛乳房炎的发生,对于提高牛群健康状态、保障和提高动物福利,对于提升乳制品的安全性和质量都是非常必要的。因此,开发一种能够普遍应用于奶牛的疫苗就显得尤为重要,而基因工程重组疫苗,是达成此目的的最有前景的研究方向之一。由于奶牛乳房炎病因复杂、病原种类繁多且具有很强的地域分布特性,单一菌株疫苗对于特定的牛群能够取得较好的预防效果,但对于不同地区病原种类不同的牛群,不但免疫过程费时费力、难以取得满意的预防效果,还会因为注射疫苗时的应激反应而影响牛群的产奶量。简单地将多种病原灭活混合,则容易导致免疫剂量过大而炎症反应严重,或是抗原含量不足,免疫效果不够理想的僵局。而基因工程亚单位疫苗的出现,使得这一看似无从下手的难题有了新的希望:针对病原种类较多的事实,可以考虑重点选择同一病原的几个关键性的保护性抗原,进而将多种病原的保护性抗原组合在一起,最终以一种多价、多联重组亚单位疫苗的形式出现,有望解决免疫剂量过大的难题,也可以解决由于抗原量不足而难以诱导产生有效的保护力的问题。本研究将传统的生化鉴定与分子生物学技术鉴定相结合,对甘肃及周边地区采集的乳房炎乳样进行病原菌的分离和鉴定;通过分子生物学技术,分析分离菌株的毒力基因和抗药基因的频率分布;采用微量肉汤稀释法分析分离菌株对临床常用抗生素的敏感情况;应用基因工程技术,构建分离菌株的真核表达载体;经转染人乳腺癌细胞表达及蛋白纯化后,对Balb/c小鼠进行免疫和攻毒试验,评价重组ClfA蛋白疫苗和DNA疫苗对Balb/c小鼠的免疫保护效果。对甘肃及周边地区采集的380份乳样进行病原分离鉴定,主要得到金黄色葡萄球菌(Staphylococcus aureus)45株、肠球菌(Enterococcus spp.)60株、大肠杆菌(Escherichia coli)13株、哈弗尼亚细菌(Hafnia)9株、链球菌(Streptococcus spp.)8株。采用微量肉汤稀释法,分析金黄色葡萄球菌对10种常用抗生素的耐药情况;其中,高于80%的金黄色葡萄球菌对环丙沙星、庆大霉素、卡那霉素和氯霉素均保持敏感,而对青霉素、氨苄西林、万古霉素具有了耐受性,此外,42%的菌株对对四环素具有耐受性。肠球菌对青霉素普遍耐受,有50%肠球菌和22%的肠球菌能够耐受高浓度的庆大霉素(500μg/ml)和链霉素(1000μg/ml),绝大部分菌株对氨苄青霉素、万古霉素和四环素敏感;分离获得的大肠杆菌中对四环素的耐受程度较高(6/13),对其余测试的抗生素基本保持敏感。采用PCR技术筛选金黄色葡萄球菌毒力基因携带情况,其中,金黄色葡萄球菌对ClfA、FnbA、hlb、hld、hlg、seb检出率较高,检出率依次为91%、88%、94%、91%、76%和64%;agr、lukS/E/M、hla、edin、eta、etb、tst和sea/c/d/e/g/i/j/n/o/m均未检测到。成功构建了金黄色葡萄球菌ClfA-pcDNA 3.1 V5-His B、肠球菌esp-pcDNA3.1 V5-His B、大肠杆菌LPS-pcDNA 3.1 V5-His B和金黄色葡萄球菌hlb-pcDNA3.1 V5-His B 4个真核表达载体。重组ClfA表达载体在MCF-7细胞中成功表达,经western blotting验证具有与天然蛋白产物相似的抗原特性。动物免疫保护试验中,重组ClfA蛋白免疫组小鼠的白细胞介素、免疫球蛋白和干扰素水平均显著高于对照组,表明重组蛋白能够给受免疫动物提供足够的保护力,符合良好的疫苗靶标特性,具有进一步开发应用的潜力。上述研究从实际出发,为深入研究和开发奶牛乳房炎病原菌多价、多联基因工程重组亚单位疫苗提供理论和实践材料。
[Abstract]:Mastitis is one of the major causes of dairy industry economic losses. After understanding and research for many years, has established a relatively complete system of prevention and control; however, in animal husbandry is relatively backward developing countries, because of the disease prevention and control theory and practice is relatively backward, the disease caused by the economic loss is still very serious. And prevent mastitis in dairy cows, cattle to improve health, protect and improve animal welfare, is necessary for the safety and quality of dairy products. Therefore, the development of a vaccine can be widely used in dairy cows is particularly important, and the recombinant gene engineering vaccine, is one of the research directions for this purpose the most promising because of mastitis etiology is complex, many kinds of pathogens and has a strong geographical characteristics of the distribution, the single strain vaccine for specific Cattle can obtain good preventive effect, but for different regions different pathogenic species of cattle, not only the immune process is time consuming, it is difficult to obtain satisfactory results but also because of prevention, response to stress vaccination and affect milk production of cattle. Simply a variety of pathogenic inactivated mixed, can easily lead to excessive immune dose while severe inflammation, or lack of immune antigen content, the effect is not ideal and deadlock. Genetic engineering subunit vaccine, a new hope to make this seemingly impossible for many pathogenic species can be considered the key facts, choose the same pathogen several key protective antigen, the combination of protective antigens of multiple pathogens together, and ultimately to a multivalent, multiple recombinant subunit vaccine appears in the form of immune dose is expected to solve the problem of the large can In order to solve the stress protective antigen is not sufficient to induce effective problems. The traditional biochemical identification and molecular biology identification technology combined with the acquisition of Gansu and the surrounding areas of the mastitis milk samples for isolation and identification of pathogens; by molecular biological technology, analysis of the frequency distribution of virulence genes of isolates the drug resistance gene; analysis of sensitivity of isolates to antibiotics by using broth microdilution method; gene engineering technique was used to construct the eukaryotic expression vector were isolated by expression of breast cancer cells; transfection and protein purification, immunity and infection test of Balb/c mice to evaluate the effect of immune protection Balb/c mouse ClfA recombinant protein vaccine and DNA vaccine. The pathogen isolation and identification of the acquisition of Gansu and the surrounding areas of the 380 milk samples, mainly by the golden yellow grape ball Bacteria (Staphylococcus aureus) and 45 strains of Enterococcus (Enterococcus spp.) and 60 strains of Escherichia coli (Escherichia coli) 13 strains of bacteria (Hafnia), Harvard, 9 strains, 8 strains of Streptococcus (Streptococcus spp.). By using the broth microdilution method, analysis of 10 kinds of antibiotic resistant Staphylococcus aureus; among them, more than 80% of the Staphylococcus aureus to ciprofloxacin, gentamicin, kanamycin and chloramphenicol were sensitive to penicillin, ampicillin, vancomycin and, with tolerance, in addition, 42% strains was tolerant to penicillin of Enterococcus to the city of Victoria. Generally well tolerated, 50% and 22% to the Enterococcus Enterococcus in high concentration tolerance to gentamicin (500 g/ml) and streptomycin (1000 g/ml), most of the strains sensitive to ampicillin, vancomycin and tetracycline; isolated Escherichia coli with tolerance to tetracycline High (6/13), for the rest of the antibiotics tested remained sensitive. Screening of Staphylococcus aureus virulence genes, which adopts PCR technology, Staphylococcus aureus ClfA, FnbA, HLB, HLD, HLG, SEB detection rate is higher, the incidence rate was 91%, 88%, 94%, 91%, 76% and 64% agr; lukS/E/M, HLA, Edin, ETA, ETB, TST, and sea/c/d/e/g/i/j/n/o/m were not detected. The successful construction of Staphylococcus aureus ClfA-pcDNA 3.1 V5-His B esp-pcDNA3.1 V5-His B, Enterococcus, Escherichia coli LPS-pcDNA 3.1 V5-His B and Staphylococcus aureus hlb-pcDNA3.1 V5-His B 4 eukaryotic expression vector. The recombinant expression vector in ClfA MCF-7 cells successfully expressed antigen characteristics similar to natural protein with Western blotting. Verify the animal immune protection test, interleukin ClfA recombinant protein immunized mice, immune globulin and interferon levels were significantly Than that of control group, showed that the recombinant protein to the immune animal protection provides enough, in line with the characteristics of a good vaccine targets, with the further development and application potential. The research from the actual situation, for further research and development of dairy cow mastitis pathogen multivalent, multiple genetic engineering subunit vaccine to provide theoretical and practical material.

【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.61

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