【摘要】：人冠狀病毒(HCoV)是人類重要的呼吸道病原體,特別是近年來呼吸窘迫綜合征冠狀病毒(SARS-COV)和中東呼吸綜合征冠狀病毒(MERS.CoV)相關(guān)疫情的暴發(fā)流行,更使其重要性日益受到重視。目前已知的HCoV主要包括α屬的NL63和229E以及p屬的OC43、HKU1、SARS-COV和MERS-COV6種,但是其抗原特性和在人群中的流行傳播情況尚未闡明。鑒此,本研究建立一套競爭性ELISA方法,分析了在HCoV感染過程中表達量最高、血清學(xué)診斷最常用的核衣殼蛋白(N蛋白)之間的抗原性和血清學(xué)關(guān)系,并通過對其抗體進行檢測分析6種HCoV在人群中的流行傳播情況。 首先利用原核表達系統(tǒng),對6種HCoV的N蛋白進行表達,并且通過陽離子交換和Ni2+-金屬螯合層析對重組N蛋白進行了純化,其純度可達到90%以上。然后,從正常人血清樣本中篩選出陽性和陰性參比血清。對6種HCoV的N蛋白進行了競爭性ELISA分析,確定6種HCoV N蛋白之間的交叉反應(yīng)關(guān)系,結(jié)果顯示亞屬內(nèi)(NL63和229E、OC43和HKU1)N蛋白之間存在交叉反應(yīng),但屬間未見交叉反應(yīng)。 利用HCoV N蛋白建立了競爭性ELISA檢測方法,以避免交叉反應(yīng)造成的干擾。在此基礎(chǔ)上,對采集自健康人群的血清樣本進行6種HCoV抗體檢測,結(jié)果顯示在受檢血清樣本中未檢測出SARS-CoV和MERS-CoV抗體,NL63、229E、OC43和HKU1抗體在人群中普遍存在,0-0.5歲組兒童血清流行率較低,分別為12.9%、0、12.9%和3.2%,但隨年齡的增加而上升,2-5歲時分別達到39%、18.1%、46.2%和17%,至15-44歲年齡組達到高峰,分別為67.1%、50.8%、70.8%和25.6%,之后隨年齡增加而降低,≥60歲年齡組分別降至25.2%、10.7%、40.3%和14.5%,NL63和OC43的血清流行率明顯高于229E和HKU1的血清流行率,提示4種常見HCoV在人群中的感染和流行水平存在差異。 為了揭示血清IgG抗體水平與急性期感染的關(guān)聯(lián)性,進一步對呼吸道樣本PCR檢測陽性和陰性病例對應(yīng)的血清樣本進行了HCoV N蛋白血清IgG抗體水平的分析,結(jié)果顯示PCR陰性樣本的血清IgG的陽性率明顯高于PCR陽性樣本的血清IgG的陽性率,提示N蛋白抗體水平可能與HCoV感染呈負(fù)相關(guān)。 綜上,本研究提示,NL63和229E、OC43和HKU1N蛋白之間存在交叉反應(yīng),利用N蛋白對HCoV的特異性N抗體檢測應(yīng)考慮亞屬內(nèi)抗原交叉反應(yīng),而通用性抗體檢測則應(yīng)考慮同時包含不同屬的抗原；在正常人群中,不同HCoV的感染率存在明顯差異,以NL63和OC43的流行為主,沒有檢測到SARS-COV和MERS-COV的感染,提示我國仍需繼續(xù)加強防控措施力度；體內(nèi)N蛋白IgG抗體水平可能與HCoV感染呈負(fù)相關(guān)。本研究為進一步深入了解HCoV感染的免疫學(xué)反應(yīng)特征、診斷試劑的研發(fā)及評估HCoV在我國的流行傳播情況提供了基礎(chǔ)。
[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 偽 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，

本文編號：2310710