人冠状病毒N蛋白之间的抗原性关系及健康人群抗体血清流行率调查研究

发布时间：2018-11-04 18:03

【摘要】：人冠状病毒(HCoV)是人类重要的呼吸道病原体,特别是近年来呼吸窘迫综合征冠状病毒(SARS-COV)和中东呼吸综合征冠状病毒(MERS.CoV)相关疫情的暴发流行,更使其重要性日益受到重视。目前已知的HCoV主要包括α属的NL63和229E以及p属的OC43、HKU1、SARS-COV和MERS-COV6种,但是其抗原特性和在人群中的流行传播情况尚未阐明。鉴此,本研究建立一套竞争性ELISA方法,分析了在HCoV感染过程中表达量最高、血清学诊断最常用的核衣壳蛋白(N蛋白)之间的抗原性和血清学关系,并通过对其抗体进行检测分析6种HCoV在人群中的流行传播情况。 首先利用原核表达系统,对6种HCoV的N蛋白进行表达,并且通过阳离子交换和Ni2+-金属螯合层析对重组N蛋白进行了纯化,其纯度可达到90%以上。然后,从正常人血清样本中筛选出阳性和阴性参比血清。对6种HCoV的N蛋白进行了竞争性ELISA分析,确定6种HCoV N蛋白之间的交叉反应关系,结果显示亚属内(NL63和229E、OC43和HKU1)N蛋白之间存在交叉反应,但属间未见交叉反应。 利用HCoV N蛋白建立了竞争性ELISA检测方法,以避免交叉反应造成的干扰。在此基础上,对采集自健康人群的血清样本进行6种HCoV抗体检测,结果显示在受检血清样本中未检测出SARS-CoV和MERS-CoV抗体,NL63、229E、OC43和HKU1抗体在人群中普遍存在,0-0.5岁组儿童血清流行率较低,分别为12.9%、0、12.9%和3.2%,但随年龄的增加而上升,2-5岁时分别达到39%、18.1%、46.2%和17%,至15-44岁年龄组达到高峰,分别为67.1%、50.8%、70.8%和25.6%,之后随年龄增加而降低,≥60岁年龄组分别降至25.2%、10.7%、40.3%和14.5%,NL63和OC43的血清流行率明显高于229E和HKU1的血清流行率,提示4种常见HCoV在人群中的感染和流行水平存在差异。 为了揭示血清IgG抗体水平与急性期感染的关联性,进一步对呼吸道样本PCR检测阳性和阴性病例对应的血清样本进行了HCoV N蛋白血清IgG抗体水平的分析,结果显示PCR阴性样本的血清IgG的阳性率明显高于PCR阳性样本的血清IgG的阳性率,提示N蛋白抗体水平可能与HCoV感染呈负相关。 综上,本研究提示,NL63和229E、OC43和HKU1N蛋白之间存在交叉反应,利用N蛋白对HCoV的特异性N抗体检测应考虑亚属内抗原交叉反应,而通用性抗体检测则应考虑同时包含不同属的抗原；在正常人群中,不同HCoV的感染率存在明显差异,以NL63和OC43的流行为主,没有检测到SARS-COV和MERS-COV的感染,提示我国仍需继续加强防控措施力度；体内N蛋白IgG抗体水平可能与HCoV感染呈负相关。本研究为进一步深入了解HCoV感染的免疫学反应特征、诊断试剂的研发及评估HCoV在我国的流行传播情况提供了基础。
[Abstract]:Human coronavirus (HCoV) is an important human respiratory pathogen, especially the outbreak of respiratory distress syndrome coronavirus (SARS-COV) and Middle East respiratory syndrome coronavirus (MERS.CoV) in recent years. It makes its importance more and more important. At present, the known HCoV mainly includes 伪 genus NL63 and 229e species and p genus OC43,HKU1,SARS-COV and MERS-COV6 species, but its antigenic characteristics and epidemic transmission in the population have not been elucidated. Therefore, a set of competitive ELISA methods was established to analyze the antigenicity and serological relationship between the nucleocapsid protein (N protein), which is the most frequently expressed protein in the process of HCoV infection, and the most commonly used nucleocapsid protein (N protein) in serological diagnosis. The prevalence and transmission of 6 kinds of HCoV in the population were analyzed by detecting their antibodies. At first, six kinds of N proteins of HCoV were expressed by prokaryotic expression system, and purified by cationic exchange and Ni2 metal chelation chromatography. The purity of the recombinant N protein was over 90%. Positive and negative reference sera were then screened from normal human serum samples. Six kinds of HCoV N proteins were analyzed by competitive ELISA, and the cross reaction relationship between 6 HCoV N proteins was determined. The results showed that there was cross reaction between subgenus (NL63 and 229EOC43 and HKU1) N protein, but there was no cross reaction between genera. HCoV N protein was used to establish a competitive ELISA assay to avoid interference caused by cross reaction. On this basis, six kinds of HCoV antibodies were detected in the serum samples collected from healthy people. The results showed that no SARS-CoV and MERS-CoV antibodies were detected in the tested serum samples, and NL63229E,OC43 and HKU1 antibodies were prevalent in the population. The seroprevalence rate of children aged 0-0.5 years was lower, 12.9% and 3.2%, respectively, but it increased with age, reaching 3918.1% and 17.2% at 2-5 years old, respectively. The age group aged 15-44 reached a peak of 67.1% and 25.6%, respectively, and then decreased with the increase of age. The group of 鈮，

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