抗牛结核分枝杆菌VHH抗体库的构建与筛选

发布时间：2018-03-26 14:59

  本文选题：牛结核分枝杆菌　切入点：VHH抗体　出处：《宁夏大学》2017年硕士论文

【摘要】：牛结核病(bovine tuberculosis)是一种死亡率极高的慢性人畜共患传染病,对公共卫生具有极大的威胁。1882年罗伯特·科赫发现了结核病发病主要是因为感染了结核分枝杆菌(M.tuberculosis,MTB),其中一定比例都是牛结核分枝杆菌引起的。牛结核分枝杆菌抗干旱抗低温,具有较强的耐药性,可以在恶劣的环境下生存,对牛结核病的防治造成了困难。1993年,比利时科学家Hamers发现骆驼体内存在一种天然缺失轻链的重链抗体,仅由其可变区组成的抗体称为单域抗体,又称为VHH抗体。VHH抗体具有高亲和力、高稳定性、强组织穿透性、高效表达等优点。因此构建抗牛结核分枝杆菌VHH抗体库,并利用噬菌体展示技术和蛋白芯片对接技术获得高表达、特异性强的VHH抗体,为诊断与治疗结核病奠定基础。本文的主要研究结果如下:1.牛结核分枝杆菌Ag85B蛋白的表达、纯化。采用BL21(DE3)(pET28a-ag85B)重组菌株,经表达、纯化、复性,获得具有活性的高浓度Ag85B蛋白。2.抗牛结核分枝杆菌VHH抗体库的构建及鉴定。实验使用BCG免疫骆驼,从骆驼外周血中分离淋巴细胞提取总RNA并反转录为cDNA,利用巢式PCR扩增VHH抗体基因片段。将目的基因片段和pCANTAB5e载体用Not Ⅰ和Sfi Ⅰ进行双酶切,用T4连接酶进行连接,转化进TG1大肠杆菌内,构建VHH抗体初级文库。对建立的VHH抗体库进行鉴定,文库库容为7.35×106。随机挑选24个单菌落进行菌液PCR,结果表明文库阳性率为91.7%。通过DNAMAN分析氨基酸序列,氨基酸序列同源性为66.48%。说明构建获得的VHH抗体库多样性丰富,库容大小足以满足特异性抗体的筛选。3.抗牛结核分枝杆菌Ag85B特异性VHH抗体的筛选。以Ag85B蛋白为抗原,利用噬菌体表面展示技术的原理,使用M13K07辅助噬菌体进行“吸附-洗脱-扩增”的淘选。经过三轮淘选后富集度达到102。以Ag85B蛋白为抗原通过蛋白芯片互作的方法随机挑取大量单克隆进行检测,筛选到对Ag85B蛋白具有高度特异性的VHH抗体。本研究利用BCG免疫骆驼,提取总RNA,构建VHH抗体库;以Ag85B对抗体文库进行三轮亲和筛选;利用蛋白芯片互作技术筛选到具有高亲和力的抗Ag85B的VHH抗体,为进一步探讨VHH抗体在结核病的诊断与治疗的研究过程中奠定了基础。
[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，

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