本文選題：奶牛乳腺炎 + 金黃色葡萄球菌��； 參考：《甘肅農業(yè)大學》2015年碩士論文

【摘要】：奶牛乳腺炎是阻礙奶牛養(yǎng)殖業(yè)發(fā)展的主要原因之一,而金黃色葡萄球菌是主要的致病菌之一,且對于不同的地區(qū),患病奶樣所分離到金黃色葡萄球菌差異較大。為查明甘肅及周邊地縣由金黃色葡萄球菌引起的奶牛乳腺炎的情況,并研制相關的基因工程疫苗,分別從甘肅秦王川、青海民和、寧夏吳忠采集380份奶樣。通過金黃色葡萄球菌耐高鹽這一特點對這批奶樣進行初步篩選,得到41株疑似金黃色葡萄球菌;再通過過氧化氫酶發(fā)酵、甘露醇發(fā)酵、三糖和Baird Parker平板等傳統(tǒng)方法對金黃色葡萄球菌進行分離鑒定,獲得37株疑似菌株;結合分子生物學方法鑒定Clf A基因,最后確定有35株為金黃色葡萄球菌的病原菌株。選取MD20菌株提取的DNA為模板,擴增ClfA基因擴增產(chǎn)物連接Trans T1感受態(tài)細胞精確測序,結果顯示,擴增長度為1168bp,與已報道的Clf A因子的基因序列(Gene Bank:HQ_424292)同源性為98%。用HindⅢ、XhoⅠ兩種酶對pGEMT-ClfA和pcDNA3.1-HisB雙酶切,并回收目的片段。后將純化后的目的片段用T4DNA連接酶于水浴鍋中16℃連接過夜后,轉化至感受態(tài)T1細胞,測序結果和菌液PCR鑒定顯示,本實驗已成功構建了真核表達載體pcDNA3.1B-Clf A。將載體pcDNA3.1B-ClfA穩(wěn)定轉染至人的乳腺癌細胞,傳代篩選至細胞穩(wěn)定,經(jīng)過trizol法提取RNA,反轉錄進行檢測,收集保存轉染成功細胞。Western-blot檢測外源蛋白的表達,分子量約為39KDa。本研究為后續(xù)的免疫動物實驗做了基礎工作,對金黃色葡萄球菌性奶牛乳腺炎的防控具有指導意義,同時也為動物之間病源互相傳播的研究提供研究依據(jù)。
[Abstract]:Cow mastitis is one of the main reasons to hinder the development of dairy cattle breeding industry, and Staphylococcus aureus is one of the main pathogenic bacteria, and for different regions, there is a great difference in Staphylococcus aureus isolated from sick milk samples. In order to find out the situation of cow mastitis caused by Staphylococcus aureus in Gansu and its surrounding counties, and to develop the related genetic engineering vaccine, 380 milk samples were collected from Qinwangchuan, Qinghai Minhe and Ningxia Wu Zhong, respectively. According to the characteristics of high salt tolerance of Staphylococcus aureus, 41 strains of suspected Staphylococcus aureus were obtained by screening the milk samples, and then by catalase fermentation and mannitol fermentation. Staphylococcus aureus was isolated from Staphylococcus aureus by traditional methods, such as trisaccharide and Baird Parker plate, 37 suspected strains were obtained, and 35 strains of Staphylococcus aureus were identified by molecular biological method. The DNA extracted from MD20 strain was selected as template to amplify the ClfA gene amplification product to connect Trans T1 competent cells. The results showed that the length of amplification was 1168 BP, which was 98% homology with gene Bank: HQs 424292 of Clf A factor. PGEMT-ClfA and pcDNA3.1-HisB were digested with Hind 鈪，

本文編號：1951940