甲型H1N1流感病毒（2009）HA蛋白的原核表达及酶免检测研究

发布时间：2018-10-29 15:31

【摘要】：2009年春季,一种新型甲型H1N1流感病毒,在短时间内迅速蔓延至全球,给人类的生命健康造成了极大的威胁,给各国带来了巨大的经济损失。本研究立项之初,国内外暂无针对此次甲型H1N1流感(2009)的商品化试剂盒,人群对此新型流感病毒普遍易感。因此,建立一种方便、快捷、灵敏的检测方法对于尽早地作出诊断和治疗、防止疫情的大流行具有重大意义。 根据病毒核蛋白和基质蛋白抗原性的不同,流感病毒可以分为甲、乙、丙三型。甲型流感病毒根据其表面糖蛋白血凝素(Hemagglutinin,HA)和神经氨酸酶的不同,又可以分为各种亚型。甲型流感病毒的8个基因节段共编码11种蛋白,其中第4节段RNA编码的HA蛋白是病毒表面最主要的糖蛋白,具有较强的免疫原性,可以激发体液免疫和细胞免疫,诱导机体产生针对流感病毒的中和抗体,是决定病毒毒力的主要因素。为了给甲型H1N1流感(2009)的及早诊断和防治提供依据,本论文选择HA蛋白作为研究对象,采用原核表达系统表达目的蛋白,将由其免疫获得的单抗用于甲型H1N1流感病毒(2009)酶免检测方法的探索。主要的研究内容由两部分组成: 1. HA抗原的表达:根据GenBank公布的甲型H1N1流感病毒(2009)血凝素基因序列,人工合成HA基因,构建原核表达载体pET-30 Xa/LIC-HA。阳性质粒转化大肠杆菌BL21(DE3),在37℃,0.1mM IPTG条件下诱导表达。重组蛋白采用镍离子亲和层析法进行纯化。对表达产物进行SDS-PAGE分析和Western blot检测。结果表明,HA基因获得表达,产物以包涵体形式存在,相对分子量约62kD,具有较好的免疫反应性。 2.甲型H1N1流感(2009)双抗体夹心检测方法的初步建立:以基因工程表达的HA重组蛋白作免疫原制备单抗,选择其中的两株配对单抗建立甲型H1N1流感病毒(2009)HA抗原双抗体夹心ELISA法,并对该方法的特异性、灵敏度、精密性和稳定性进行评估。结果表明,该方法能特异性地检出甲型H1N1流感病毒(2009),而与H1N2、H3N2、H5N1和乙型流感病毒无交叉反应;对HA抗原的最低检出限达到1ng/mL,且具有较好的精密性和稳定性。
[Abstract]:In the spring of 2009, a new type of influenza A (H1N1) virus spread to the whole world in a short period of time, which caused great threat to human life and health, and brought huge economic losses to all countries. At the beginning of this study, there was no commercial kit for H1N1 influenza (2009) at home and abroad, and the population was generally susceptible to the new influenza virus. Therefore, it is of great significance to establish a convenient, fast and sensitive detection method for diagnosis and treatment as early as possible to prevent the epidemic. According to the antigenicity of viral nucleoprotein and matrix protein, influenza virus can be classified into three types: type A, B and C. Influenza A virus can be divided into subtypes according to its surface glycoprotein hemagglutinin (Hemagglutinin,HA) and neuraminidase. The 8 gene segments of influenza A virus encode 11 kinds of proteins, of which the HA protein encoded by the fourth segment of RNA is the most important glycoprotein on the surface of the virus. It has strong immunogenicity and can stimulate humoral and cellular immunity. Inducing the body to produce neutralizing antibody against influenza virus is the main factor to determine the virulence of the virus. In order to provide basis for early diagnosis and prevention and treatment of H1N1 influenza A (2009), HA protein was selected as the research object, and the target protein was expressed by prokaryotic expression system. The monoclonal antibody was used to detect the influenza A H1N1 virus (2009) by enzyme immunoassay. The main research content consists of two parts: 1. Expression of HA antigen: according to the hemagglutinin gene sequence of A H1N1 influenza virus (2009) published by GenBank, the HA gene was synthesized and the prokaryotic expression vector pET-30 Xa/LIC-HA. was constructed. The positive plasmid was transformed into Escherichia coli BL21 (DE3) and was induced to express at 37 鈩，

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