SARS和MERS中和抗体假病毒检测方法的建立
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摘要
严重急性呼吸综合征冠状病毒(SARS-CoV)和中东呼吸综合征冠状病毒(MERS-CoV)均属冠状病毒科(Coronaviridae),能够引起人类严重疾病,特别是SARS-CoV可以导致严重急性呼吸综合征,而且容易造成暴发式流行,引起社会恐慌。本研究利用含有SARS-CoV和MERS-CoV棘突蛋白(Spike protein,S)基因的表达质粒pcD-NA3.1-SS、pcDNA3.1-MS,成功构建了高滴度假病毒,在此基础上通过对病毒接种量进行优化,建立了稳定可靠的假病毒中和抗体检测方法,并通过检测SARS恢复期病人血清以及免疫后小鼠血清对方法的特异性、稳定性以及重复性进行分析,充分证明了该方法是有效的血清中和抗体的检测手段。本研究成功构建了不依赖于BSL-3级生物安全条件的SARS和MERS假病毒,不仅可应用于血清中和抗体检测,还为后续单克隆抗体及抗病毒药物筛选和评价、疫苗研发及病毒感染机制研究等奠定了基础。
The severe acute respiratory syndrome coronavirus(SARS-CoV)and Middle East respiratory syndrome coronavirus(MERS-CoV)belong to Coronaviridae,a family of enveloped,positive-sense,single-stranded RNA viruses that can cause serious human diseases. In particular,an outbreak of SARS caused by SARS-CoV can lead to social panic. We constructed stable pseudoviruses using pcDNA3.1-SS and pcDNA3.1-MS containing the S genes of SARS-CoV and MERS-CoV. On this basis,a method for detecting neutralizing antibodies was established by optimizing the inoculation amount of pseudoviruses. The specificity,stability and repeatability of this method were evaluated using the sera of patients convalescing from SARS and sera from im-munized mice. Results demonstrated that this method could be used to detect neutralizing antibodies in sera.Construction of SARS and MERS pseudoviruses independent from a Biological Safety Level-3 laboratory could be applied for detection of neutralizing antibodies from sera. Such construction could also lay a foundation for subsequent screening and evaluation of monoclonal antibodies and antiviral drugs,vaccine development,and research on the mechanism of viral infection.
The severe acute respiratory syndrome coronavirus(SARS-CoV)and Middle East respiratory syndrome coronavirus(MERS-CoV)belong to Coronaviridae,a family of enveloped,positive-sense,single-stranded RNA viruses that can cause serious human diseases. In particular,an outbreak of SARS caused by SARS-CoV can lead to social panic. We constructed stable pseudoviruses using pcDNA3.1-SS and pcDNA3.1-MS containing the S genes of SARS-CoV and MERS-CoV. On this basis,a method for detecting neutralizing antibodies was established by optimizing the inoculation amount of pseudoviruses. The specificity,stability and repeatability of this method were evaluated using the sera of patients convalescing from SARS and sera from im-munized mice. Results demonstrated that this method could be used to detect neutralizing antibodies in sera.Construction of SARS and MERS pseudoviruses independent from a Biological Safety Level-3 laboratory could be applied for detection of neutralizing antibodies from sera. Such construction could also lay a foundation for subsequent screening and evaluation of monoclonal antibodies and antiviral drugs,vaccine development,and research on the mechanism of viral infection.
引文
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[15]Fukushi S,Mizutani T,M,Kurane I,Taguchi F,Tashiro M,Morikawa S. Evaluation of a novel vesicular stomatitis virus pseudotype-based assay for detection of neutralizing antibody responses to SARS-CoV[J]. J Med Virol,2010,78(12):1509-1512.
[16]李琳,郭彦,赵光宇,于虹,孙世惠,潘婷,唐健,周育森,樊卫平.中东呼吸综合征冠状病毒假病毒系统的建立及其在中和抗体检测中的应用[J].生物技术通讯,2014,25(2):194-197.
[17]闫克夏,谭文杰,张相民,王慧娟,李岩,阮力.SARS-CoV假病毒中和试验技术的建立及评价[J].病毒学报,2007,23(6):440-446. DOI:10.13242/j.cnki.bingduxue-bao.001855
[18]Lu L,Liu Q,Zhu Y,Chan K H,Qin L,Li Y,Wang Q,Chan J F,Du L,Yu F,Ma C,Ye S,Yuen K Y,Zhang R,Jiang S. Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor[J]. Nat Commun,2014,5:3067.
[19]Han D P,Kim H G,Kim Y B,Poon L L,Cho M W.Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein[J]. Virolo-gy,2004,326(1):140-149.
[20]Zhao G,Du L,Ma C,Li Y,Li L,Poon V K,Wang L,Yu F,Zheng B J,Jiang S,Zhou Y. A safe and con-venient pseudovirus-based inhibition assay to detect neu-tralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV[J].Virol J,2013,10:266.
[2] Zaki A M,Van Boheemen S,Bestebroer T M,Oster-haus A D,Fouchier R A. Isolation of a novel coronavi-rus from a man with pneumonia in Saudi Arabia[J]. N Engl J Med,2012,367(19):1814-1820.
[3] Centers for Disease C,Prevention. Severe respiratory ill-ness associated with a novel coronavirus--Saudi Arabia and Qatar,2012[J]. MMWR Morb Mortal Wkly Rep,2012,61(40):820.
[4] Parren P W,Burton D R. The antiviral activity of anti-bodies in vitro and in vivo[J]. Adv Immunol,2001,77:195-262.
[5]毛乃颖,张燕,朱贞,崔爱利,刘中华,杨建国,严汉民,蒋小泓,张建,阮力.检测人血清中SARS冠状病毒IgG抗体的ELISA方法建立及其应用[J].病毒学报,2004,20(1):73-78. DOI:10.13242/j. cnki. bingduxue-bao.001604
[6]王慧娟,王文玲,蓝佳明,宋铁,甄维,周为民,柯昌文,武桂珍,谭文杰.基于全病毒颗粒的中东呼吸综合征冠状病毒抗体检测方法的建立[J].中华微生物学和免疫学杂志,2016,36(2):93-97.
[7]万卓越,鄢心革,张欣,李晖,李杰,刁丽梅,林锦炎.IFA检测SARS CoV的应用研究[J].中华微生物学和免疫学杂志,2003,23(12):939-940.
[8]安祺,董关木,刘文雪,杨立宏,孔艳.SARS冠状病毒空斑技术的建立和应用[J].中国生物制品学杂志,2004,17(6):400-402.
[9]王文玲,黄保英,王慧娟,赵莉,陆柔剑,朱娜,邓瑶,谭文杰.基于Avicel-结晶紫染色空斑的中东呼吸综合征冠状病毒感染滴定与中和抗体检测方法[J].中华微生物学和免疫学杂志,2017,37(1):62-67.
[10]Ksiazek T G,Erdman D,Goldsmith C S,Zaki S R,Peret T,Emery S,Tong S,Urbani C,Comer J A,Lim W,Rollin P E,Dowell S F,Ling A E,Humphrey C D,Shieh W J,Guarner J,Paddock C D,Rota P,Fields B,Derisi J,Yang J Y,Cox N,Hughes J M,Leduc J W,Bellini W J,Anderson L J,Group S W. A novel coronavirus associated with severe acute respirato-ry syndrome[J]. N Engl J Med,2003,348(20):1953-1966.
[11]Li Q,Liu Q,Huang W,Li X,Wang Y. Current status on the development of pseudoviruses for enveloped virus-es[J/OL]. Rev Med Virol,2017,28(1).
[12]Klasse P J,Sattentau Q J. Occupancy and mechanism in antibody-mediated neutralization of animal viruses[J]. J Gen Virol,2002,83(9):2091-2108.
[13]Zhu Z,Chakraborti S,He Y,Roberts A,Sheahan T,Xiao X,Hensley L E,Prabakaran P,Rockx B,Sidorov I A,Corti D,Vogel L,Feng Y,Kim J O,Wang L F,Baric R,Lanzavecchia A,Curtis K M,Nabel G J,Sub-barao K,Jiang S,Dimitrov D S. Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies[J]. Proc Natl Acad Sci,2007,104(29):12123-12128.
[14]Corti D,Zhao J,Pedotti M,Simonelli L,Agnihothram S,Fett C,Fernandez-Rodriguez B,Foglierini M,Agat-ic G,Vanzetta F,Gopal R,Langrish C J,Barrett N A,Sallusto F,Baric R S,Varani L,Zambon M,Perlman S,Lanzavecchia A. Prophylactic and postexposure effi-cacy of a potent human monoclonal antibody against MERS coronavirus[J]. Proc Natl Acad Sci,2015,112(33):10473-10478.
[15]Fukushi S,Mizutani T,M,Kurane I,Taguchi F,Tashiro M,Morikawa S. Evaluation of a novel vesicular stomatitis virus pseudotype-based assay for detection of neutralizing antibody responses to SARS-CoV[J]. J Med Virol,2010,78(12):1509-1512.
[16]李琳,郭彦,赵光宇,于虹,孙世惠,潘婷,唐健,周育森,樊卫平.中东呼吸综合征冠状病毒假病毒系统的建立及其在中和抗体检测中的应用[J].生物技术通讯,2014,25(2):194-197.
[17]闫克夏,谭文杰,张相民,王慧娟,李岩,阮力.SARS-CoV假病毒中和试验技术的建立及评价[J].病毒学报,2007,23(6):440-446. DOI:10.13242/j.cnki.bingduxue-bao.001855
[18]Lu L,Liu Q,Zhu Y,Chan K H,Qin L,Li Y,Wang Q,Chan J F,Du L,Yu F,Ma C,Ye S,Yuen K Y,Zhang R,Jiang S. Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor[J]. Nat Commun,2014,5:3067.
[19]Han D P,Kim H G,Kim Y B,Poon L L,Cho M W.Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein[J]. Virolo-gy,2004,326(1):140-149.
[20]Zhao G,Du L,Ma C,Li Y,Li L,Poon V K,Wang L,Yu F,Zheng B J,Jiang S,Zhou Y. A safe and con-venient pseudovirus-based inhibition assay to detect neu-tralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV[J].Virol J,2013,10:266.
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