广州管圆线虫半乳凝素基因的克隆表达、蛋白纯化及免疫反应性研究

发布时间：2018-01-12 03:38

本文关键词：广州管圆线虫半乳凝素基因的克隆表达、蛋白纯化及免疫反应性研究　出处：《第一军医大学》2007年硕士论文　论文类型：学位论文

【摘要】： 广州管圆线虫病(Angiostrongyliasis)是由广州管圆线虫(Angiostrongylus cantonensis)感染而引起的。作为一种新发的食源性传染性疾病，广州管圆线虫病在全球的流行都呈现出上升的趋势，对人类的威胁也越来越大。广州管圆线虫主要侵犯中枢神经系统，引起嗜酸性粒细胞增多性脑膜炎或脑膜脑炎。患该病后，绝大多数患者有头痛、肌肉痛、恶心、呕吐、感觉异常、触摸痛、发热、畏光、视力模糊、嗜睡、颈硬等症状。其中以头痛最突出，患者自诉“头痛欲裂”或忍受不了；全身疼痛、皮肤过敏的现象也比较严重；个别病人还会出现精神病样症状。我国是广州管圆线虫病的主要流行区之一，近几年的发病数逐渐增多，甚至出现了爆发流行。如何有效地防治广州管圆线虫病已成为亟待解决的重要问题。在广州管圆线虫病的防治工作中，，诊断是关键。诊断明确，治疗及时，则病人的转归一般良好。但目前国内外都还缺乏敏感特异、能产业化生产、标准化的诊断试剂盒。针对目前这个难点问题，本论文进行了以下研究：一、广州管圆线虫半乳凝素基因的获取及生物信息学分析应用EST策略，我们从广州管圆线虫脑期幼虫cDNA文库中克隆了半乳凝素基因(AcGal)的全长cDNA序列，并已在GenBank中登录(登录号为DQ384534)。AcGal基因与秀丽杆线虫半乳凝素-10高度同源。从保守结构域的搜索结果可知，AcGal具有半乳凝素结构域，该结构域能专一性地与β-半乳糖苷结合，如乳糖，而不需要金属离子的活化。半乳凝素以二聚体或三聚体结构域存在。二、广州管圆线虫半乳凝素基因在大肠杆菌中的表达及免疫反应性分析 PCR扩增AcGal cDNA全长序列后克隆入pET32a(+)载体进行诱导表达，包涵体经洗涤、变性、逐步透析复性，浓缩后进行聚丙烯酰胺凝胶电泳及免疫印迹。AcGal基因在大肠杆菌中以包涵体的形式表达，表达产物与感染广州管圆线虫的大鼠血清有明显的免疫反应性，具有诊断用抗原的应用前景。综上所述，本研究利用本室构建的广州管圆线虫幼虫cDNA文库首次筛选克隆了广州管圆线虫半乳凝素的全长cDNA序列，并通过进一步的研究发现其重组蛋白是一种具有诊断潜力的抗原。
[Abstract]:Angiostrongyliasis Angiostrongylus cantonensis (Angiostrongylus cantonensis). Caused by infection. As a new foodborne infectious disease. The global prevalence of Angiostrongylus cantonensis is on the rise and the threat to human is increasing. Angiostrongylus cantonensis mainly invades the central nervous system. Causes eosinophilic meningitis or meningoencephalitis. After the disease, the vast majority of patients have headache, muscle pain, nausea, vomiting, sensory abnormalities, touch pain, fever, photophobia, blurred vision, sleepiness. Neck hard and other symptoms. One of the most prominent headache, the patient complained of "headache to crack" or can not endure; Systemic pain, skin allergy phenomenon is more serious; Individual patients will also have psychotic symptoms. China is one of the main endemic areas of Angiostrongylus cantonensis. In recent years, the incidence of the disease has gradually increased. Even an outbreak has occurred. How to effectively control Angiostrongylus cantonensis has become an important problem to be solved. In the prevention and control of Angiostrongylus cantonensis, diagnosis is the key. If the diagnosis is clear and the treatment is timely, the outcome of the patients is generally good. But at present, there is still a lack of sensitivity and specificity at home and abroad, and can be industrialized production. Standardized diagnostic kit. In view of this difficult problem, this paper has carried out the following research: 1. Acquisition and bioinformatics analysis of galactin gene of Angiostrongylus cantonensis Using the EST strategy, we cloned the full-length cDNA sequence of galactin gene from the cDNA library of the brain larvae of Angiostrongylus cantonensis. It has been registered in GenBank (accession number is DQ384534).AcGal gene and highly homologous to galactin -10 of nematode elegans). The search results of conserved domain can be seen. AcGal has a galactin domain which specifically binds to 尾 -galactoside such as lactose and does not require activation by metal ions. Galactin exists as a dimer or trimer domain. 2. Expression and immunoreactivity analysis of galactin gene of Angiostrongylus cantonensis in Escherichia coli The full-length AcGal cDNA sequence was amplified by PCR and cloned into pET32a () vector to induce the expression. The inclusion bodies were washed, denatured, and gradually dialysis renatured. After concentration, polyacrylamide gel electrophoresis and Western blot. AcGal gene was expressed as inclusion body in Escherichia coli. The expressed product has obvious immunoreactivity with the serum of rats infected with Angiostrongylus cantonensis and has the prospect of application of diagnostic antigen. In conclusion, the full-length cDNA sequence of galactin from Angiostrongylus cantonensis larvae was screened and cloned for the first time by using the cDNA library constructed in our laboratory. Through further study, it was found that the recombinant protein was a potential antigen for diagnosis.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R383

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