發(fā)布時(shí)間：2018-03-20 07:18

  本文選題：亞洲璃眼蜱　切入點(diǎn)：組胺　出處：《上海師范大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：蜱是一種常見(jiàn)的吸血體外寄生蟲(chóng),給畜牧業(yè)和人類(lèi)的健康帶來(lái)了嚴(yán)重的影響。蜱的寄生會(huì)引起宿主失血、不安和皮膚損傷,同時(shí)在叮咬過(guò)程中蜱可以傳播多種疾病。蜱叮咬可以誘發(fā)宿主產(chǎn)生炎癥反應(yīng)、凝血反應(yīng)和免疫反應(yīng)等,為了克服宿主的這些反應(yīng),蜱的唾液腺通過(guò)分泌一些生物大分子干擾宿主的反應(yīng),如抗凝血分子、免疫抑制分子和抗炎癥分子等,這些分子有成為治療血凝相關(guān)疾病,腫瘤,以及自身免疫性疾病的藥物的潛力,其中蜱組胺結(jié)合蛋白(Histamine binding protein)就是這些分子中的一種。蜱組胺結(jié)合蛋白是由蜱的唾液腺分泌的具有特異性結(jié)合組胺的一類(lèi)功能性蛋白的總稱(chēng),是蜱的重要抗炎癥分子之一。亞洲璃眼蜱隸屬于硬蜱科璃眼屬,為三宿主蜱,廣泛分布于我國(guó)的西北部地區(qū)(新疆、內(nèi)蒙、寧夏)以及中亞地區(qū)。本實(shí)驗(yàn)室成功從亞洲璃眼蜱唾液腺中克隆表達(dá)出組胺結(jié)合蛋白(Histamine binding protein of Hyalomma asiaticum,Ha HBP)。為了進(jìn)一步深入研究Ha HBP的功能特點(diǎn),在已有的實(shí)驗(yàn)數(shù)據(jù)的基礎(chǔ)上對(duì)亞洲璃眼蜱唾液腺中的Ha HBP進(jìn)行了重組表達(dá)及生物學(xué)功能分析。首先,對(duì)實(shí)驗(yàn)室保存的p GEX-4T-1-Ha HBP/BL21重組質(zhì)粒的菌種進(jìn)行活化,經(jīng)擴(kuò)大培養(yǎng),16℃低溫,1M IPTG誘導(dǎo),GST樹(shù)脂親和層析純化,獲得了純度較高的Ha HBP-GST重組蛋白。其次,將編碼Ha HBP的基因片段克隆至真核表達(dá)載體p Fast Bac HTa,將重組質(zhì)粒轉(zhuǎn)化至DH10Bac感受態(tài)細(xì)胞中,構(gòu)建重組桿粒r Bac-Ha HBP,轉(zhuǎn)染至sf9昆蟲(chóng)細(xì)胞,進(jìn)行重組蛋白的表達(dá)純化。經(jīng)SDS-PAGE、Western blot和IFA檢測(cè)結(jié)果表明,Ha HBP在Bac-to-Bac桿狀病毒系統(tǒng)中穩(wěn)定表達(dá),重組蛋白大小與預(yù)期一致。將原核表達(dá)和真核表達(dá)的Ha HBP重組蛋白在不同濃度梯度的條件下與HRP-histamine共同孵育,進(jìn)行組胺結(jié)合實(shí)驗(yàn),結(jié)果表明,兩種表達(dá)系統(tǒng)所表達(dá)的重組蛋白都具有組胺結(jié)合的能力。最后,分別通過(guò)涂抹和滴鼻給藥的方法探究Ha HBP對(duì)二甲苯誘發(fā)小鼠耳腫模型和OVA誘發(fā)的小鼠哮喘模型的作用效果,實(shí)驗(yàn)結(jié)果表明,Ha HBP對(duì)小鼠的耳腫和哮喘都具有一定的抑制作用。綜上所述,本研究對(duì)組胺結(jié)合蛋白(HBP)的表達(dá)和生物學(xué)功能進(jìn)行了初步探究,為研究新型蜱源抗組胺藥物提供了新的思路。
[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，

本文編號(hào)：1638038