本文選題：奶牛乳腺炎 + 金黃色葡萄球菌��； 參考：《甘肅農(nóng)業(yè)大學(xué)》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(duì)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(guān)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(guò)金黃色葡萄球菌耐高鹽這一特點(diǎn)對(duì)這批奶樣進(jìn)行初步篩選,得到41株疑似金黃色葡萄球菌;再通過(guò)過(guò)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(duì)金黃色葡萄球菌進(jìn)行分離鑒定,獲得37株疑似菌株;結(jié)合分子生物學(xué)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(kuò)增ClfA基因擴(kuò)增產(chǎn)物連接Trans T1感受態(tài)細(xì)胞精確測(cè)序,結(jié)果顯示,擴(kuò)增長(zhǎng)度為1168bp,與已報(bào)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(duì)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(guò)夜后,轉(zhuǎn)化至感受態(tài)T1細(xì)胞,測(cè)序結(jié)果和菌液PCR鑒定顯示,本實(shí)驗(yàn)已成功構(gòu)建了真核表達(dá)載體pcDNA3.1B-Clf A。將載體pcDNA3.1B-ClfA穩(wěn)定轉(zhuǎn)染至人的乳腺癌細(xì)胞,傳代篩選至細(xì)胞穩(wěn)定,經(jīng)過(guò)trizol法提取RNA,反轉(zhuǎn)錄進(jìn)行檢測(cè),收集保存轉(zhuǎn)染成功細(xì)胞。Western-blot檢測(cè)外源蛋白的表達(dá),分子量約為39KDa。本研究為后續(xù)的免疫動(dòng)物實(shí)驗(yàn)做了基礎(chǔ)工作,對(duì)金黃色葡萄球菌性奶牛乳腺炎的防控具有指導(dǎo)意義,同時(shí)也為動(dòng)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(hào)：1951940