甲型H1N1流感病毒（2009）HA蛋白的原核表達(dá)及酶免檢測(cè)研究
發(fā)布時(shí)間：2018-10-29 15:31
【摘要】：2009年春季,一種新型甲型H1N1流感病毒,在短時(shí)間內(nèi)迅速蔓延至全球,給人類(lèi)的生命健康造成了極大的威脅,給各國(guó)帶來(lái)了巨大的經(jīng)濟(jì)損失。本研究立項(xiàng)之初,國(guó)內(nèi)外暫無(wú)針對(duì)此次甲型H1N1流感(2009)的商品化試劑盒,人群對(duì)此新型流感病毒普遍易感。因此,建立一種方便、快捷、靈敏的檢測(cè)方法對(duì)于盡早地作出診斷和治療、防止疫情的大流行具有重大意義。 根據(jù)病毒核蛋白和基質(zhì)蛋白抗原性的不同,流感病毒可以分為甲、乙、丙三型。甲型流感病毒根據(jù)其表面糖蛋白血凝素(Hemagglutinin,HA)和神經(jīng)氨酸酶的不同,又可以分為各種亞型。甲型流感病毒的8個(gè)基因節(jié)段共編碼11種蛋白,其中第4節(jié)段RNA編碼的HA蛋白是病毒表面最主要的糖蛋白,具有較強(qiáng)的免疫原性,可以激發(fā)體液免疫和細(xì)胞免疫,誘導(dǎo)機(jī)體產(chǎn)生針對(duì)流感病毒的中和抗體,是決定病毒毒力的主要因素。為了給甲型H1N1流感(2009)的及早診斷和防治提供依據(jù),本論文選擇HA蛋白作為研究對(duì)象,采用原核表達(dá)系統(tǒng)表達(dá)目的蛋白,將由其免疫獲得的單抗用于甲型H1N1流感病毒(2009)酶免檢測(cè)方法的探索。主要的研究?jī)?nèi)容由兩部分組成: 1. HA抗原的表達(dá):根據(jù)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,構(gòu)建原核表達(dá)載體pET-30 Xa/LIC-HA。陽(yáng)性質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3),在37℃,0.1mM IPTG條件下誘導(dǎo)表達(dá)。重組蛋白采用鎳離子親和層析法進(jìn)行純化。對(duì)表達(dá)產(chǎn)物進(jìn)行SDS-PAGE分析和Western blot檢測(cè)。結(jié)果表明,HA基因獲得表達(dá),產(chǎn)物以包涵體形式存在,相對(duì)分子量約62kD,具有較好的免疫反應(yīng)性。 2.甲型H1N1流感(2009)雙抗體夾心檢測(cè)方法的初步建立:以基因工程表達(dá)的HA重組蛋白作免疫原制備單抗,選擇其中的兩株配對(duì)單抗建立甲型H1N1流感病毒(2009)HA抗原雙抗體夾心ELISA法,并對(duì)該方法的特異性、靈敏度、精密性和穩(wěn)定性進(jìn)行評(píng)估。結(jié)果表明,該方法能特異性地檢出甲型H1N1流感病毒(2009),而與H1N2、H3N2、H5N1和乙型流感病毒無(wú)交叉反應(yīng);對(duì)HA抗原的最低檢出限達(dá)到1ng/mL,且具有較好的精密性和穩(wěn)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，
本文編號(hào)：2298088
[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，
本文編號(hào)：2298088
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