本文選題：牛結(jié)核分枝桿菌　切入點(diǎn)：VHH抗體　出處：《寧夏大學(xué)》2017年碩士論文

【摘要】：牛結(jié)核病(bovine tuberculosis)是一種死亡率極高的慢性人畜共患傳染病,對(duì)公共衛(wèi)生具有極大的威脅。1882年羅伯特·科赫發(fā)現(xiàn)了結(jié)核病發(fā)病主要是因?yàn)楦腥玖私Y(jié)核分枝桿菌(M.tuberculosis,MTB),其中一定比例都是牛結(jié)核分枝桿菌引起的。牛結(jié)核分枝桿菌抗干旱抗低溫,具有較強(qiáng)的耐藥性,可以在惡劣的環(huán)境下生存,對(duì)牛結(jié)核病的防治造成了困難。1993年,比利時(shí)科學(xué)家Hamers發(fā)現(xiàn)駱駝體內(nèi)存在一種天然缺失輕鏈的重鏈抗體,僅由其可變區(qū)組成的抗體稱為單域抗體,又稱為VHH抗體。VHH抗體具有高親和力、高穩(wěn)定性、強(qiáng)組織穿透性、高效表達(dá)等優(yōu)點(diǎn)。因此構(gòu)建抗牛結(jié)核分枝桿菌VHH抗體庫(kù),并利用噬菌體展示技術(shù)和蛋白芯片對(duì)接技術(shù)獲得高表達(dá)、特異性強(qiáng)的VHH抗體,為診斷與治療結(jié)核病奠定基礎(chǔ)。本文的主要研究結(jié)果如下:1.牛結(jié)核分枝桿菌Ag85B蛋白的表達(dá)、純化。采用BL21(DE3)(pET28a-ag85B)重組菌株,經(jīng)表達(dá)、純化、復(fù)性,獲得具有活性的高濃度Ag85B蛋白。2.抗牛結(jié)核分枝桿菌VHH抗體庫(kù)的構(gòu)建及鑒定。實(shí)驗(yàn)使用BCG免疫駱駝,從駱駝外周血中分離淋巴細(xì)胞提取總RNA并反轉(zhuǎn)錄為cDNA,利用巢式PCR擴(kuò)增VHH抗體基因片段。將目的基因片段和pCANTAB5e載體用Not Ⅰ和Sfi Ⅰ進(jìn)行雙酶切,用T4連接酶進(jìn)行連接,轉(zhuǎn)化進(jìn)TG1大腸桿菌內(nèi),構(gòu)建VHH抗體初級(jí)文庫(kù)。對(duì)建立的VHH抗體庫(kù)進(jìn)行鑒定,文庫(kù)庫(kù)容為7.35×106。隨機(jī)挑選24個(gè)單菌落進(jìn)行菌液PCR,結(jié)果表明文庫(kù)陽(yáng)性率為91.7%。通過(guò)DNAMAN分析氨基酸序列,氨基酸序列同源性為66.48%。說(shuō)明構(gòu)建獲得的VHH抗體庫(kù)多樣性豐富,庫(kù)容大小足以滿足特異性抗體的篩選。3.抗牛結(jié)核分枝桿菌Ag85B特異性VHH抗體的篩選。以Ag85B蛋白為抗原,利用噬菌體表面展示技術(shù)的原理,使用M13K07輔助噬菌體進(jìn)行“吸附-洗脫-擴(kuò)增”的淘選。經(jīng)過(guò)三輪淘選后富集度達(dá)到102。以Ag85B蛋白為抗原通過(guò)蛋白芯片互作的方法隨機(jī)挑取大量單克隆進(jìn)行檢測(cè),篩選到對(duì)Ag85B蛋白具有高度特異性的VHH抗體。本研究利用BCG免疫駱駝,提取總RNA,構(gòu)建VHH抗體庫(kù);以Ag85B對(duì)抗體文庫(kù)進(jìn)行三輪親和篩選;利用蛋白芯片互作技術(shù)篩選到具有高親和力的抗Ag85B的VHH抗體,為進(jìn)一步探討VHH抗體在結(jié)核病的診斷與治療的研究過(guò)程中奠定了基礎(chǔ)。
[Abstract]:Bovine tuberculosis tuberculosisis a chronic zoonotic disease with a high mortality rate. A great threat to public health. In 1882, Robert Koch discovered that tuberculosis was mainly caused by infection with M. tuberculosisus MTBN, a certain proportion of which was caused by Mycobacterium bovis. Mycobacterium bovis is resistant to drought and hypothermia. Having strong drug resistance, they can survive in harsh conditions, making it difficult to prevent and cure bovine tuberculosis. In 1993, Belgian scientist Hamers found a naturally absent heavy chain antibody in camels. The antibody composed of only its variable region is called single domain antibody, also called VHH antibody. VHH antibody has the advantages of high affinity, high stability, strong tissue penetration and high expression. Therefore, the VHH antibody library against Mycobacterium bovis is constructed. The highly expressed and specific VHH antibody was obtained by phage display technique and protein chip docking technique, which laid a foundation for the diagnosis and treatment of tuberculosis. The main results of this study are as follows: 1. The expression of Ag85B protein in Mycobacterium bovis. The recombinant strain BL21DE3, pET28a-ag85B, was expressed, purified and renatured to obtain high concentration Ag85B protein. 2. Construction and identification of VHH antibody library against Mycobacterium bovis. BCG was used to immunize camels. Total RNA was extracted from camel peripheral blood lymphocytes and reversely transcribed into cDNA. VHH antibody gene fragment was amplified by nested PCR. The target gene fragment and pCANTAB5e vector were digested with Not 鈪，

本文編號(hào)：1668346