本文選題：無(wú)乳鏈球菌 + Sip蛋白��； 參考：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：奶牛乳腺炎是奶牛最常見(jiàn)的疾病之一,影響奶牛的產(chǎn)奶量和牛奶品質(zhì),嚴(yán)重的可導(dǎo)致奶牛泌乳功能喪失甚至被迫淘汰,大大降低奶牛養(yǎng)殖的經(jīng)濟(jì)效益,給養(yǎng)牛業(yè)造成巨大的經(jīng)濟(jì)損失。同時(shí)病原微生物可通過(guò)污染的奶制品而感染人,對(duì)人類(lèi)健康造成危害。無(wú)乳鏈球菌是奶牛乳腺炎的主要病原之一,無(wú)乳鏈球菌感染雖不引起乳房明顯的膿腫或纖維化,但是可以導(dǎo)致腺體感染,造成產(chǎn)奶量持續(xù)減少。其表面免疫相關(guān)蛋白(Sip)暴露在細(xì)菌的表面,在多種血清型的無(wú)乳鏈球菌菌株中均有表達(dá),是細(xì)菌重要的黏附和定植因子之一,具有較好的免疫保護(hù)作用和良好的抗原性,是無(wú)乳鏈球菌重要的候選疫苗抗原之一。九種不同血清型菌株Sip蛋白氨基酸序列比較結(jié)果顯示此蛋白質(zhì)高度保守。因此,利用此蛋白開(kāi)發(fā)一個(gè)迅速有效的檢測(cè)方法監(jiān)控?zé)o乳鏈球菌感染,提高乳制品的質(zhì)量和安全,具有重要的應(yīng)用價(jià)值。本研究參照Gen Bank公布的牛源無(wú)乳鏈球菌Sip基因序列(AF151361)設(shè)計(jì)一對(duì)特異性引物,成功構(gòu)建p GEX-6p-1-Sip原核表達(dá)質(zhì)粒。經(jīng)IPTG誘導(dǎo)表達(dá)及條件優(yōu)化,成功在大腸桿菌感受態(tài)Rosetta(DE)中表達(dá)了分子量為59.8 Ku的無(wú)乳鏈球菌Sip重組蛋白,其優(yōu)化條件為終濃度1.0 mmol/L IPTG 37℃恒溫誘導(dǎo)5 h。純化的重組蛋白為其后噬菌體的篩選提供了材料。隨后,以純化的無(wú)乳鏈球菌Sip重組蛋白為靶分子,用M13噬菌體c DNA文庫(kù),進(jìn)行4輪噬菌體生物淘選。無(wú)乳鏈球菌Sip蛋白的首輪包被量為10μg/孔,此后每輪依次減半,噬菌體每輪投入量均為1.5×1011 CFU/μL。經(jīng)過(guò)噬菌體淘洗、擴(kuò)增和滴度測(cè)定,最終在總數(shù)少于100的LB/IPTG/Xgal平板上隨機(jī)挑取10個(gè)噬菌體藍(lán)斑于搖菌管中進(jìn)行4.5 h擴(kuò)增后測(cè)定其滴度。擴(kuò)增后的10個(gè)噬菌體分別進(jìn)行噬菌體與靶分子的噬菌體ELISA親和能力的測(cè)試,檢測(cè)結(jié)果顯示,所獲取的10個(gè)隨機(jī)噬菌體均與靶分子具有高度的親和能力,為ELISA方法檢測(cè)無(wú)乳鏈球菌的建立奠定了基礎(chǔ)。利用能與無(wú)乳鏈球菌Sip蛋白結(jié)合的噬菌體作為第一抗體,M13多抗作為第二抗體,建立了可快速檢測(cè)含無(wú)乳鏈球菌奶樣的間接ELISA檢測(cè)方法并進(jìn)行了條件優(yōu)化。優(yōu)化結(jié)果為:抗原包被濃度(即敏感度)為10 CFU/mL/孔,噬菌體最佳作用濃度為108 PFU/mL;抗原最佳包被方式確定為37℃包被2 h時(shí)后4℃過(guò)夜的方式;最佳封閉劑的選擇為1.0%BSA磷酸鹽緩沖液置37℃封閉3 h;噬菌體最佳孵育時(shí)間為60 min;M13多抗1:1000倍稀釋孵育45 min;酶標(biāo)抗體最佳稀釋度為1:4000,最佳孵育時(shí)間為45 min。并用所建立的間接ELISA檢測(cè)方法進(jìn)行了無(wú)乳鏈球菌敏感性、特異性和臨床樣本的檢測(cè),敏感性可檢測(cè)到10 CFU/mL。臨床樣本檢測(cè)了某牧場(chǎng)54份患奶牛乳腺炎的奶樣,檢測(cè)陽(yáng)性率達(dá)14.286%。本實(shí)驗(yàn)將噬菌體表面展示技術(shù)與ELISA檢測(cè)方法相結(jié)合,為奶牛乳腺炎無(wú)乳鏈球菌提供了簡(jiǎn)便高效的新型檢測(cè)方法。
[Abstract]:Cow mastitis is one of the most common diseases in dairy cows, which affects the milk yield and quality of cows, which can seriously lead to the loss of lactation function and even forced elimination of dairy cows, which greatly reduces the economic benefits of dairy cattle breeding. Great economic losses have been caused to the cattle industry. At the same time, pathogenic microorganisms can infect people through contaminated dairy products, which is harmful to human health. Streptococcus actinomycetes is one of the main pathogens of dairy cow mastitis. Although it does not cause abscess or fibrosis of breast, it can cause gland infection and decrease milk production. The surface immune-associated protein (Sip-Sip) is expressed on the surface of bacteria and expressed in various serotypes of Streptococcus lactis. It is one of the important adhesion and colonization factors of bacteria, and has good immune protection and antigenicity. It is one of the most important candidate vaccine antigens for Streptococcus lactobacillus. The amino acid sequence analysis of Sip protein of nine different serotype strains showed that the protein was highly conserved. Therefore, it is of great value to develop a rapid and effective method to detect streptococcus lactococcus infection and improve the quality and safety of dairy products. In this study, a pair of specific primers were designed according to the Sip gene sequence AF151361 published by Gen Bank, and the prokaryotic expression plasmid of p GEX-6p-1-Sip was successfully constructed. The recombinant protein of Streptococcus lactis Sip with molecular weight of 59.8 Ku was successfully expressed in Escherichia coli (E. coli) by IPTG induced expression and optimized conditions. The optimal conditions were as follows: 1. 0 mmol/L IPTG at 37 鈩，

本文編號(hào)：1791339