本文關(guān)鍵詞：廣州管圓線蟲半乳凝素基因的克隆表達、蛋白純化及免疫反應(yīng)性研究　出處：《第一軍醫(yī)大學》2007年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 廣州管圓線蟲 半乳凝索 克隆 表達 純化 免疫反應(yīng)性

【摘要】： 廣州管圓線蟲病(Angiostrongyliasis)是由廣州管圓線蟲(Angiostrongylus cantonensis)感染而引起的。作為一種新發(fā)的食源性傳染性疾病，廣州管圓線蟲病在全球的流行都呈現(xiàn)出上升的趨勢，對人類的威脅也越來越大。廣州管圓線蟲主要侵犯中樞神經(jīng)系統(tǒng)，引起嗜酸性粒細胞增多性腦膜炎或腦膜腦炎�；荚摬『�，絕大多數(shù)患者有頭痛、肌肉痛、惡心、嘔吐、感覺異常、觸摸痛、發(fā)熱、畏光、視力模糊、嗜睡、頸硬等癥狀。其中以頭痛最突出，患者自訴“頭痛欲裂”或忍受不了；全身疼痛、皮膚過敏的現(xiàn)象也比較嚴重；個別病人還會出現(xiàn)精神病樣癥狀。我國是廣州管圓線蟲病的主要流行區(qū)之一，近幾年的發(fā)病數(shù)逐漸增多，甚至出現(xiàn)了爆發(fā)流行。如何有效地防治廣州管圓線蟲病已成為亟待解決的重要問題。 在廣州管圓線蟲病的防治工作中，，診斷是關(guān)鍵。診斷明確，治療及時，則病人的轉(zhuǎn)歸一般良好。但目前國內(nèi)外都還缺乏敏感特異、能產(chǎn)業(yè)化生產(chǎn)、標準化的診斷試劑盒。針對目前這個難點問題，本論文進行了以下研究： 一、廣州管圓線蟲半乳凝素基因的獲取及生物信息學分析 應(yīng)用EST策略，我們從廣州管圓線蟲腦期幼蟲cDNA文庫中克隆了半乳凝素基因(AcGal)的全長cDNA序列，并已在GenBank中登錄(登錄號為DQ384534)。AcGal基因與秀麗桿線蟲半乳凝素-10高度同源。從保守結(jié)構(gòu)域的搜索結(jié)果可知，AcGal具有半乳凝素結(jié)構(gòu)域，該結(jié)構(gòu)域能專一性地與β-半乳糖苷結(jié)合，如乳糖，而不需要金屬離子的活化。半乳凝素以二聚體或三聚體結(jié)構(gòu)域存在。 二、廣州管圓線蟲半乳凝素基因在大腸桿菌中的表達及免疫反應(yīng)性分析 PCR擴增AcGal cDNA全長序列后克隆入pET32a(+)載體進行誘導(dǎo)表達，包涵體經(jīng)洗滌、變性、逐步透析復(fù)性，濃縮后進行聚丙烯酰胺凝膠電泳及免疫印跡。AcGal基因在大腸桿菌中以包涵體的形式表達，表達產(chǎn)物與感染廣州管圓線蟲的大鼠血清有明顯的免疫反應(yīng)性，具有診斷用抗原的應(yīng)用前景。 綜上所述，本研究利用本室構(gòu)建的廣州管圓線蟲幼蟲cDNA文庫首次篩選克隆了廣州管圓線蟲半乳凝素的全長cDNA序列，并通過進一步的研究發(fā)現(xiàn)其重組蛋白是一種具有診斷潛力的抗原。
[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.
【學位授予單位】：第一軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R383

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