本文選題：奶牛乳房炎　切入點：病原　出處：《甘肅農業(yè)大學》2016年博士論文　論文類型：學位論文

【摘要】：奶牛乳房炎是造成奶牛飼養(yǎng)行業(yè)經濟損失的重要原因之一。經過多年的認識和研究,已經建立起來了比較完備的預防和控制體系;但是,在畜牧業(yè)相對落后的發(fā)展中國家,由于疾病防控理論和實踐相對落后,該病造成的經濟損失依然非常嚴重。減少和杜絕奶牛乳房炎的發(fā)生,對于提高牛群健康狀態(tài)、保障和提高動物福利,對于提升乳制品的安全性和質量都是非常必要的。因此,開發(fā)一種能夠普遍應用于奶牛的疫苗就顯得尤為重要,而基因工程重組疫苗,是達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復雜、病原種類繁多且具有很強的地域分布特性,單一菌株疫苗對于特定的牛群能夠取得較好的預防效果,但對于不同地區(qū)病原種類不同的牛群,不但免疫過程費時費力、難以取得滿意的預防效果,還會因為注射疫苗時的應激反應而影響牛群的產奶量。簡單地將多種病原滅活混合,則容易導致免疫劑量過大而炎癥反應嚴重,或是抗原含量不足,免疫效果不夠理想的僵局。而基因工程亞單位疫苗的出現(xiàn),使得這一看似無從下手的難題有了新的希望:針對病原種類較多的事實,可以考慮重點選擇同一病原的幾個關鍵性的保護性抗原,進而將多種病原的保護性抗原組合在一起,最終以一種多價、多聯(lián)重組亞單位疫苗的形式出現(xiàn),有望解決免疫劑量過大的難題,也可以解決由于抗原量不足而難以誘導產生有效的保護力的問題。本研究將傳統(tǒng)的生化鑒定與分子生物學技術鑒定相結合,對甘肅及周邊地區(qū)采集的乳房炎乳樣進行病原菌的分離和鑒定;通過分子生物學技術,分析分離菌株的毒力基因和抗藥基因的頻率分布;采用微量肉湯稀釋法分析分離菌株對臨床常用抗生素的敏感情況;應用基因工程技術,構建分離菌株的真核表達載體;經轉染人乳腺癌細胞表達及蛋白純化后,對Balb/c小鼠進行免疫和攻毒試驗,評價重組ClfA蛋白疫苗和DNA疫苗對Balb/c小鼠的免疫保護效果。對甘肅及周邊地區(qū)采集的380份乳樣進行病原分離鑒定,主要得到金黃色葡萄球菌(Staphylococcus aureus)45株、腸球菌(Enterococcus spp.)60株、大腸桿菌(Escherichia coli)13株、哈弗尼亞細菌(Hafnia)9株、鏈球菌(Streptococcus spp.)8株。采用微量肉湯稀釋法,分析金黃色葡萄球菌對10種常用抗生素的耐藥情況;其中,高于80%的金黃色葡萄球菌對環(huán)丙沙星、慶大霉素、卡那霉素和氯霉素均保持敏感,而對青霉素、氨芐西林、萬古霉素具有了耐受性,此外,42%的菌株對對四環(huán)素具有耐受性。腸球菌對青霉素普遍耐受,有50%腸球菌和22%的腸球菌能夠耐受高濃度的慶大霉素(500μg/ml)和鏈霉素(1000μg/ml),絕大部分菌株對氨芐青霉素、萬古霉素和四環(huán)素敏感;分離獲得的大腸桿菌中對四環(huán)素的耐受程度較高(6/13),對其余測試的抗生素基本保持敏感。采用PCR技術篩選金黃色葡萄球菌毒力基因攜帶情況,其中,金黃色葡萄球菌對ClfA、FnbA、hlb、hld、hlg、seb檢出率較高,檢出率依次為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均未檢測到。成功構建了金黃色葡萄球菌ClfA-pcDNA 3.1 V5-His B、腸球菌esp-pcDNA3.1 V5-His B、大腸桿菌LPS-pcDNA 3.1 V5-His B和金黃色葡萄球菌hlb-pcDNA3.1 V5-His B 4個真核表達載體。重組ClfA表達載體在MCF-7細胞中成功表達,經western blotting驗證具有與天然蛋白產物相似的抗原特性。動物免疫保護試驗中,重組ClfA蛋白免疫組小鼠的白細胞介素、免疫球蛋白和干擾素水平均顯著高于對照組,表明重組蛋白能夠給受免疫動物提供足夠的保護力,符合良好的疫苗靶標特性,具有進一步開發(fā)應用的潛力。上述研究從實際出發(fā),為深入研究和開發(fā)奶牛乳房炎病原菌多價、多聯(lián)基因工程重組亞單位疫苗提供理論和實踐材料。
[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.

【學位授予單位】：甘肅農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S852.61

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