应用跨膜区置换的血凝素蛋白建立H7N9禽流感病毒抗体间接ELISA检测方法

发布时间：2018-02-13 00:14

本文关键词： 流感病毒 HN亚型间接ELISA 血凝素蛋白跨膜区　出处：《微生物学通报》2017年12期 　论文类型：期刊论文

【摘要】：【目的】由于H7N9禽流感病毒能够感染鸡,并且已经变异成了高致病性毒株,因此,鸡群中H7N9禽流感疫苗的免疫是一个趋势,而鸡群免疫后抗体检测方法的建立也十分必要。本研究旨在建立一种灵敏、高效、高通量的鸡群H7N9亚型禽流感病毒抗体间接酶联免疫吸附试验(ELISA)检测方法。【方法】通过昆虫杆状病毒表达系统分别表达属于W1、W2-A和W2-B分支H7N9流感病毒的3种野生型血凝素(HA)蛋白,以及跨膜区(TM)置换为H3 HA TM的W2-B分支HA蛋白(H7-53TM)。4种HA蛋白经过离子交换层析纯化后作为抗原,通过ELISA检测H7N9禽流感病毒抗体。【结果】ELISA特异性、敏感性和重复性试验结果显示,跨膜区置换主要影响HA蛋白ELISA检测的重复性,以H7-53TM为抗原的ELISA方法具有较好的重复性,其批内和批间变异系数小于10%,然而3种野生型HA蛋白与部分血清反应批内和批间变异系数大于10%,重复性较差,因此选择H7-53TM蛋白作为ELISA包被抗原。通过受试者工作特征曲线(ROC曲线)分析,以H7-53TM为抗原的ELISA能够精准地区分H7N9亚型流感病毒抗体阳性和阴性血清。通过相关性分析,该ELISA方法与134份鸡血清HI试验结果具有显著强相关性(r=0.854 6,P0.000 1),并且与3个分支疫苗株免疫血清的HI试验结果也具有显著相关性(r0.5,P0.05)。【结论】跨膜区置换能够提高HA蛋白抗原检测H7N9禽流感病毒抗体的重复性,并应用跨膜区置换的HA蛋白建立了一种能够检测不同分支疫苗株免疫的H7N9亚型禽流感病毒抗体间接ELISA检测方法。
[Abstract]:[objective] since H7N9 avian influenza virus can infect chickens and has mutated into highly pathogenic strain, immunization of H7N9 avian influenza vaccine in chickens is a trend. It is also necessary to establish a method for the detection of antibodies in chickens after immunization. The purpose of this study was to establish a sensitive and efficient method for the detection of antibodies in chickens. High throughput Elisa was used to detect H7N9 subtype avian influenza virus antibody in chickens. [methods] three wild type hemagglutinin (HAA) proteins belonging to W1W2-A and W2-B branch H7N9 influenza virus were expressed by baculovirus expression system. The HA protein H7-53TM.4 was purified by ion exchange chromatography and used as antigen to detect H7N9 avian influenza virus antibody by ELISA. [results] the specificity, sensitivity and reproducibility of H7N9 avian influenza virus were detected by ELISA specificity, sensitivity and reproducibility test. Transmembrane region replacement mainly affected the reproducibility of ELISA detection of HA protein. The ELISA method with H7-53TM as antigen had good reproducibility. The coefficient of variation within and between batches was less than 10, however, the coefficient of variation between the three wild type HA proteins and partial serum was greater than 10, and the repeatability was poor. Therefore, H7-53TM protein was selected as ELISA coated antigen. By using the operating characteristic curve of the subjects, ELISA with H7-53TM as antigen could accurately distinguish positive and negative sera against H7N9 subtype influenza virus. The ELISA method has a significant correlation with the HI test results of 134 chicken serum samples, and also with the HI test results of three branch vaccine strains. [conclusion] Transmembrane region replacement can improve HA protein. The reproducibility of detection of H7N9 avian influenza virus antibody by antigens, An indirect ELISA detection method of H7N9 subtype avian influenza virus antibody was established by using the HA protein transmembrane region to detect the immune response of different branch vaccine strains to Avian Influenza virus (Avian Influenza virus).
【作者单位】：中山大学生命科学学院有害生物控制与资源利用国家重点实验室;
【基金】：广州市科技计划项目(No.201504010025) 广东省农业厅H7N9流感联合攻关项目(粤财农[2014]380号)~~
【分类号】：S858.31

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