本文選題：牛外周血單個核細(xì)胞 + 布氏桿菌病　； 參考：《延邊大學(xué)》2015年碩士論文

【摘要】：布氏桿菌病(Brucellosis)是一種人畜共患傳染病。19世紀(jì)末期被人們發(fā)現(xiàn)并廣泛研究,迄今為止已經(jīng)有100多年的歷史。布氏桿菌病是我國規(guī)定的乙類動物疫病,也是目前嚴(yán)重威脅養(yǎng)牛業(yè)發(fā)展和人類健康的重要疾病之一。酵母雙雜交系統(tǒng)是用來研究蛋白質(zhì)與蛋白質(zhì)、蛋白質(zhì)與小分子之間相互作用的實驗技術(shù)。是通過建立牛外周血單個核細(xì)胞的cDNA文庫,來篩選與牛布氏桿菌結(jié)合的受體蛋白,對布氏桿菌病的預(yù)防和控制有重要的意義。本研究預(yù)先制備牛外周血單個核細(xì)胞,采用Trizol法提取牛外周血單個核細(xì)胞的總RNA。利用美國Clontech公司的SMART技術(shù)構(gòu)建牛外周血單個核細(xì)胞的酵母雙雜交cDNA文庫。.將全部的DNA合成cDNA的前端,然后在整個反應(yīng)中加入SMART Ⅲ Oligo,目的是讓cDNA的前端與mRNA形成互補,合成雙鏈cDNA的方法是通過長距離PCR(LD-PCR)。因為雙鏈cDNA與PGADT7-Rec利用酵母細(xì)胞內(nèi)具有非常相似的重組酶活性,進(jìn)而形成具有復(fù)制活性的文庫質(zhì)粒,那么在SD/Leu培養(yǎng)板上篩選出所有克隆。所以用純化后的雙鏈cDNA與線性化質(zhì)粒PGADT7-Rec連接,轉(zhuǎn)入酵母細(xì)胞Y187感受態(tài)中,并用PEG/LiAc的方法完成轉(zhuǎn)化。再次通過稀釋的方法計算轉(zhuǎn)化率與文庫的容量大小,然后通過菌落PCR的方法檢查重組率和插入片段的大小。結(jié)果顯示,提取的牛外周血單個核細(xì)胞的總RNA濃度為1050ng/ul, OD26o/OD280為1.95,后經(jīng)1%瓊脂糖凝膠電泳分析可見28S和18S條帶。純化后雙鏈cDNA的凝膠電泳的方法操作,結(jié)果顯示片段大小在400-2 500bp之間。鑒定結(jié)果表明,本次構(gòu)建的cDNA文庫容量為4.0×108CFU/mL,文庫的重組率達(dá)到95%以上,平均插入片段的大小為700bp左右。以上實驗結(jié)果說明本研究建立的cDNA文庫的質(zhì)量非常高,達(dá)到了后續(xù)實驗的要求,為牛布氏桿菌受體的篩選提供了扎實的基礎(chǔ)與理論依據(jù)。
[Abstract]:Brucellosis (Brucellosis) is a zoonotic infectious disease. It has been widely studied in the late 19th century and has a history of more than 100 years. Brucellosis is one of the most important diseases that threaten the development of cattle industry and human health. Yeast two-hybrid system is used to study the interaction between protein and protein, protein and small molecule. CDNA library of bovine peripheral blood mononuclear cells was established to screen receptor proteins binding to Brucella bovis, which is of great significance for the prevention and control of brucellosis. In this study, bovine peripheral blood mononuclear cells (BPBMC) were prepared and extracted from bovine peripheral blood mononuclear cells (BPBMC) by Trizol method. The yeast two-hybrid cDNA library of bovine peripheral blood mononuclear cells (BPBMC) was constructed by SMART technique of Clontech Company in USA. The front end of cDNA was synthesized from all DNA and then SMART 鈪，

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