H5亚型禽流感病毒HA抗原表位多肽筛选和多肽ELISA检测抗体方法的建立

发布时间：2018-01-16 23:21

本文关键词：H5亚型禽流感病毒HA抗原表位多肽筛选和多肽ELISA检测抗体方法的建立　出处：《沈阳农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：禽流感(Avian influenza, AI)是由A型禽流感病毒(Avian influenza virus, AIV)引起的一种主要在禽类中传播的感染性疾病。AIV感染家禽后可引发呼吸道疾病,造成产蛋下降,严重时可造成100%的死亡率。近年来AIV频繁变异,使得某些突变毒株可直接感染人类并导致人死亡,严重危害公共卫生,并且造成巨大经济损失,受到密切关注。现地检测主要通过ELISA试验鸡抗血清中的AIV抗体的存在与否判断鸡群的免疫情况及是否有AIV野毒感染,目前ELISA试验的包被抗原主要为灭活的H5亚型全病毒或纯化的H5亚型HA蛋白,且ELISA试剂盒多为进口,价格高,不利该病的检测。本研究通过筛选H5亚型HA的特异性抗原表位多肽,并以此作为ELISA试验的包被抗原,建立可特异性检测H5亚型AIV HA抗体的ELISA试验方法,为生产具有自主知识产权的ELISA试剂盒打下基础。在本研究中,以H1-H15亚型流感病毒和新城疫病毒制备了灭活的免疫抗原,并将其免疫SPF鸡,获得抗H1-H15亚型流感病毒和新城疫病毒的免疫血清。将本实验室保存的H5亚型AIV (A/DuTurkey/England/N28/1973)的HA氨基酸序列作为模板,合成抗原表位多肽,每条抗原表位多肽含有15个氨基酸,并且后一条多肽与前一条重叠14个氨基酸。将合成的H5亚型HA抗原表位多肽固定于固相载体上,与已知血清反应,通过肽扫描技术,筛选出可与抗H5亚型AIV鸡血清特异性反应的4条抗原表位多肽,命名为H5-1、H5-2、H5-3、H5-4。将筛选出的H5-1抗原表位多肽包被酶联免疫吸附板,通过矩阵试验确定最佳的抗原包被量及血清样品稀释度,并对ELISA试验的抗原包被、样品稀释、二抗稀释及显色等试验条件进行优化。以最终确定的ELISA试验条件分别包被4条H5亚型HA抗原表位多肽并对H1-H15亚型禽流感病毒鸡抗血清进行检测,结果发现仅H5-1抗原表位多肽能有效的将H5亚型禽流感病毒鸡抗血清与其他亚型禽流感病毒鸡抗血清区分开,H5-2、H5-3、H5-4则不同程度的受到其他亚型禽流感病毒鸡抗血清的干扰,特异性不强。将H5-1抗原表位多肽检测新城疫病毒鸡抗血清,结果发现其不与新城疫病毒鸡抗血清发生反应。系列梯度滴定试验确定该ELISA试验方法适合检测1：200倍稀释的血清样品,符合性试验证明对于H5亚型血凝抑制价大于1的样品血清,该方法阳性检出率大于87.6%,H5亚型血凝抑制价大于4的样品血清阳性检出率98.3%。将H5-1抗原表位多肽分别从氨基端和羧基端进行截短,并将截短多肽对应的核酸片段插入原核表达载体pGEX-6p-1,诱导原核表达,通过蛋白免疫印迹(western blot)试验确定H5-1抗原表位多肽的核心基序为HNIHP。
[Abstract]:Avian influenza virus (AIA) is composed of avian influenza virus type A (Avian influenza virus). AIV) an infectious disease mainly transmitted in poultry. Infection of poultry can lead to respiratory diseases, resulting in a drop in egg laying. In recent years, AIV frequently mutates, which causes some mutant strains to infect human beings directly and lead to human death, seriously endanger public health, and cause huge economic losses. In situ, the presence of AIV antibodies in the sera of chickens tested with ELISA was used to determine the immune status of chickens and whether they had AIV field virus infection. At present, the coating antigen of ELISA test is mainly inactivated H5 subtype virus or purified H5 subtype HA protein, and the ELISA kit is mostly imported, and the price is high. In this study, the specific epitope peptides of H5 subtype HA were screened and used as the coated antigen for ELISA test. To establish a ELISA assay for the specific detection of H5 subtype AIV HA antibody, and to lay a foundation for the production of ELISA kit with independent intellectual property rights. The inactivated immune antigen of H1-H15 influenza virus and Newcastle disease virus was prepared and immunized with SPF chicken. The immune serum against H1-H15 influenza virus and Newcastle disease virus was obtained. The H5 subtype AIV (. The HA amino acid sequence of A / U Turkey / Englandr / N 28 / 1973 was used as a template. Antigenic epitope peptides were synthesized. Each epitope peptide contained 15 amino acids. The latter peptide overlapped with the former one 14 amino acids. The synthesized H5 subtype HA epitope peptide was immobilized on the solid phase carrier and reacted with the known serum. The peptide scanning technique was used. Four antigenic epitopes which could specifically react with anti-H5 subtype AIV chicken serum were selected and named H5-1H5-2H5-3. H5-4. enzyme linked immunosorbent plate (Elisa) was used to determine the best antigen envelope and the dilution of serum samples, and the antigenic envelope of ELISA test was determined by matrix test. Sample dilution. Four HA epitope peptides of H5 subtype were coated with H1-H15 avian influenza virus avian influenza virus antiserum by the final ELISA test conditions, and the antiserum of H1-H15 subtype Avian Influenza virus was injected into the chicken antiserum of H1-H15 subtype Avian Influenza virus (Avian Influenza virus). Check. The results showed that only H5N1 epitope peptide could effectively distinguish H5 subtype avian influenza virus chicken antiserum from other subtype avian influenza virus chicken antiserum. H5-4 was interfered by the chicken antiserum of other subtypes of avian influenza virus, and the specificity of H5-4 was not strong. Detection of Newcastle disease virus chicken antiserum by H5N1 epitope peptide. The results showed that it did not react with Newcastle disease virus chicken antiserum. A series of gradient titration tests confirmed that the ELISA test was suitable for the detection of 1: 200 diluted serum samples. The conformance test showed that the positive rate of this method was more than 87.6% for serum samples with H5 subtype hemagglutination inhibitory value greater than 1. The positive rate of H5 subtype hemagglutination inhibitory value was 98.3%. The H 5-1 epitope peptides were truncated from amino terminal and carboxyl terminal, respectively. Nucleic acid fragments corresponding to truncated peptides were inserted into prokaryotic expression vector pGEX-6p-1 to induce prokaryotic expression. The core motif of H 5-1 epitope peptide was identified as HNIHP by Western blot assay.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65

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