二球悬铃木花粉主要变应原编码基因的克隆与表达

发布时间：2018-02-12 02:23

本文关键词： 花粉重组变应原克隆表达　出处：《南京医科大学》2006年硕士论文　论文类型：学位论文

【摘要】：背景：近数十年以来，世界范围内各种变态反应病发病率明显增加，，尤其在工业化程度较高的发达国家，各种过敏性疾病严重影响居民生活质量，提高医疗费用，造成经济损失，已经成为世界性卫生问题。人群中约有五分之一属于过敏体质，有可能发生过敏性疾病。世界卫生组织已将此类疾病列为“21世纪重点研究和预防的疾病”。花粉是最常见的气传吸入性过敏原之一，可引起多种过敏性疾病，如过敏性鼻炎、支气管哮喘、过敏性结膜炎等。“花粉症”即季节性变应性鼻炎，又称枯草热。二球悬铃木花粉是近年来发现的我国南方地区常见的引起特应性体质者发生过敏反应的过敏原，对其变应原组分和分子生物学特点的研究开始受到研究者重视。用基因工程制备人工重组变应原具有天然变应原提取物不可比拟的优越性，国外已开始研制常见变应原的人工重组蛋白，逐步应用于临床诊断和特异性免疫治疗。目的：提取天然二球悬铃木花粉总RNA，制备其主要变应原的编码基因克隆，诱导表达人工重组蛋白，鉴定其生物学活性和免疫学特异性。方法：采集二球悬铃木花粉，Trizol法提取总RNA，逆转录为cDNA。设计其主要变应原编码基因的特异性引物，用PCR扩增目的片段。将扩增的目的片段用双酶切法与质粒pET32a连接，用CaCl_2法转入工程菌DH5α制备基因克隆。提取基因克隆导入工程菌BL21a(DE3)，碱裂解法提取
[Abstract]:Background: in recent decades, the incidence of allergy diseases has increased significantly worldwide, especially in the highly industrialized developed countries, which seriously affect the quality of life of residents and increase medical costs. Causing economic losses, it has become a worldwide health problem. About 1/5 of the population belong to allergic constitution, which may lead to allergic diseases. The World Health Organization has listed these diseases as "diseases focused on research and prevention in 21th century". Pollen is one of the most common airborne inhaled allergens, which can cause many allergic diseases, such as allergic rhinitis, bronchial asthma, allergic conjunctivitis and so on. The pollen of Suzuki mongolica is a common anaphylaxis found in southern China in recent years. The study of its allergen components and molecular biological characteristics has attracted more and more attention. The preparation of artificial recombinant allergens by genetic engineering has incomparable advantages over natural allergen extracts. Foreign countries have begun to develop artificial recombinant proteins of common allergens, which have been gradually used in clinical diagnosis and specific immunotherapy. Objective: to extract the total RNA of pollen from the natural two-ball suspension of Suzuki Suzuki, prepare the encoding gene clone of its main allergen, induce and express the artificial recombinant protein, and identify its biological activity and immunological specificity. Methods: the total RNAs were extracted by Trizol method and reverse transcripted as cDNA.The specific primers of the main allergen encoding genes were designed and amplified by PCR. The amplified fragments were linked with plasmid pET32a by double enzyme digestion. The gene clone was obtained by transferring DH5 伪 into engineering bacteria by CaCl_2 method. The gene clone was extracted and introduced into the engineering bacterium BL21a DE3, and extracted by alkali lysis method.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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