蜱组胺结合蛋白的重组表达及其生物学功能分析

发布时间：2018-03-20 07:18

  本文选题：亚洲璃眼蜱　切入点：组胺　出处：《上海师范大学》2015年硕士论文　论文类型：学位论文

【摘要】：蜱是一种常见的吸血体外寄生虫,给畜牧业和人类的健康带来了严重的影响。蜱的寄生会引起宿主失血、不安和皮肤损伤,同时在叮咬过程中蜱可以传播多种疾病。蜱叮咬可以诱发宿主产生炎症反应、凝血反应和免疫反应等,为了克服宿主的这些反应,蜱的唾液腺通过分泌一些生物大分子干扰宿主的反应,如抗凝血分子、免疫抑制分子和抗炎症分子等,这些分子有成为治疗血凝相关疾病,肿瘤,以及自身免疫性疾病的药物的潜力,其中蜱组胺结合蛋白(Histamine binding protein)就是这些分子中的一种。蜱组胺结合蛋白是由蜱的唾液腺分泌的具有特异性结合组胺的一类功能性蛋白的总称,是蜱的重要抗炎症分子之一。亚洲璃眼蜱隶属于硬蜱科璃眼属,为三宿主蜱,广泛分布于我国的西北部地区(新疆、内蒙、宁夏)以及中亚地区。本实验室成功从亚洲璃眼蜱唾液腺中克隆表达出组胺结合蛋白(Histamine binding protein of Hyalomma asiaticum,Ha HBP)。为了进一步深入研究Ha HBP的功能特点,在已有的实验数据的基础上对亚洲璃眼蜱唾液腺中的Ha HBP进行了重组表达及生物学功能分析。首先,对实验室保存的p GEX-4T-1-Ha HBP/BL21重组质粒的菌种进行活化,经扩大培养,16℃低温,1M IPTG诱导,GST树脂亲和层析纯化,获得了纯度较高的Ha HBP-GST重组蛋白。其次,将编码Ha HBP的基因片段克隆至真核表达载体p Fast Bac HTa,将重组质粒转化至DH10Bac感受态细胞中,构建重组杆粒r Bac-Ha HBP,转染至sf9昆虫细胞,进行重组蛋白的表达纯化。经SDS-PAGE、Western blot和IFA检测结果表明,Ha HBP在Bac-to-Bac杆状病毒系统中稳定表达,重组蛋白大小与预期一致。将原核表达和真核表达的Ha HBP重组蛋白在不同浓度梯度的条件下与HRP-histamine共同孵育,进行组胺结合实验,结果表明,两种表达系统所表达的重组蛋白都具有组胺结合的能力。最后,分别通过涂抹和滴鼻给药的方法探究Ha HBP对二甲苯诱发小鼠耳肿模型和OVA诱发的小鼠哮喘模型的作用效果,实验结果表明,Ha HBP对小鼠的耳肿和哮喘都具有一定的抑制作用。综上所述,本研究对组胺结合蛋白(HBP)的表达和生物学功能进行了初步探究,为研究新型蜱源抗组胺药物提供了新的思路。
[Abstract]:Tick is a common extracorporeal blood sucking parasite that has serious effects on animal husbandry and human health. Tick parasitism can cause blood loss, unease and skin damage to the host. At the same time, ticks can spread many diseases in the course of biting. Tick bites can induce inflammation, coagulation and immune response of the host, in order to overcome these reactions of the host. Ticks' salivary glands interfere with host responses by secreting biological macromolecules, such as anticoagulants, immunosuppressive molecules and anti-inflammatory molecules, which can be used to treat hemagglutination-related diseases, tumors, and so on. And the potential of drugs for autoimmune diseases, among which histamine binding protein (histamine binding protein) is one of these molecules. Tick histamine binding protein is a class of functional proteins secreted by the salivary glands of ticks that specifically bind to histamine. Acari is one of the most important anti-inflammatory molecules in ticks. It is a three-host ticks belonging to the genus Artemisia, which is widely distributed in northwest China (Xinjiang, Inner Mongolia, Inner Mongolia, Xinjiang, Inner Mongolia, China). In order to further study the functional characteristics of Ha HBP, we cloned and expressed histamine binding protein of Hyalomma asiaticum Ha HBPU from salivary glands of P. asiaticum in Ningxia and Central Asia in order to further study the functional characteristics of Ha HBP. The recombination expression and biological function of Ha HBP in salivary gland of P. asiatica were analyzed on the basis of available experimental data. Firstly, the strain of recombinant plasmid of p GEX-4T-1-Ha HBP/BL21 was activated. The recombinant protein Ha HBP-GST was purified by expanded culture at 16 鈩，

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