表达趋化因子配体13的重组A组16型柯萨奇病毒的构建
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摘要
柯萨奇病毒A组16型(Coxsackievirus A16,CV-A16)是小核糖核酸病毒科肠道病毒属病毒,是手足口病(Hand,foot and mouse disease,HFMD)的主要病原体之一。CV-A16在世界范围内广泛流行,但目前还没有有效预防CV-A16感染的疫苗上市,CV-A16疫苗的研发仍处于实验室研究阶段。本研究从改善CV-A16诱导体液免疫能力的角度出发,利用反向遗传学手段构建共表达趋化因子配体13的重组A组16型柯萨奇病毒(Coxsackievirus A16-Chemokine(C-X-C motif)ligand 13,CV-A16-CXCL13)。首先利用全基因合成方法设计合成MluⅠ-T7-5'UTR-CXCL13-VP4-VP2-NdeⅠ片段,将该片段插入pCR-XL-TOPO-CV-A16载体中,获得pCR-XL-TOPO-CV-A16-CXCL13克隆载体。将该载体线性化,体外转录出重组病毒RNA并转染细胞,最终获得包装成功的CV-A16-CXCL13重组病毒。使用PCR检测到病毒感染细胞后趋化因子配体13(Chemokine (C-X-C motif) ligand 13,CXCL13)及结构蛋白VP1在基因水平的表达;免疫印记及免疫荧光结果检测到(CXCL13与VP1蛋白水平的表达;电镜结果显示重组病毒为直径20～30nm球状颗粒;增殖曲线显示重组病毒能够在细胞中正常复制。本研究成功构建了CV-A16-CXCL13病毒,为CV-A16感染特征的研究和疫苗研发提供参考。
Coxsackievirus A16(CV-A16) is a member of the Enterovirus genus of Picornaviridae and is one of the major pathogens associated with hand, foot and mouse disease(HFMD). The CV-A16 has been epidemical in the world, but there is no vaccine to effectively prevent CV-A16 infection. The development of CV-A16 vaccine is still in the laboratory research stage. In order to promote the activation of humoral immunity induced by CV-A16,this study used reverse genetics to construct a recombinant Coxsackievirus A16(C V-A16-CXCL13)that co-expresses chemokine ligand 13. The Mlu Ⅰ-T7-5'UTR-CXCL13-VP4-VP2-Nde I fragment was designed and synthesized. The fragment was inserted into pCR-XL-TOPO-CV-A16 vector to obtain pCR-XLTOPO-CV-A16-CXCL13 clone vector. The vector was linearized, and the recombinant viral RNA was transcribed in vitro and transfected into cells. Finally, the successfully packaged CV-A16-CXCL13 recombinant virus was obtained. Expression of CXCL13 and structural protein VP1 were detected at the gene level by PCR with virus-infected cells. Immunoblotting and immunofluorescence results showed that CXCL13 and VP1 protein were simultaneously expressed in infected cells. Electron microscopy showed that the recombinant virus particles were 20～30 nm in diameter. The proliferation curve showed that the recombinant virus was able to replicate normally in the cell. This paper successfully constructed the CV-A16-CXCL13 virus, which will be useful for the study of the characteristics of CV-A16 infection and development of vaccines.
Coxsackievirus A16(CV-A16) is a member of the Enterovirus genus of Picornaviridae and is one of the major pathogens associated with hand, foot and mouse disease(HFMD). The CV-A16 has been epidemical in the world, but there is no vaccine to effectively prevent CV-A16 infection. The development of CV-A16 vaccine is still in the laboratory research stage. In order to promote the activation of humoral immunity induced by CV-A16,this study used reverse genetics to construct a recombinant Coxsackievirus A16(C V-A16-CXCL13)that co-expresses chemokine ligand 13. The Mlu Ⅰ-T7-5'UTR-CXCL13-VP4-VP2-Nde I fragment was designed and synthesized. The fragment was inserted into pCR-XL-TOPO-CV-A16 vector to obtain pCR-XLTOPO-CV-A16-CXCL13 clone vector. The vector was linearized, and the recombinant viral RNA was transcribed in vitro and transfected into cells. Finally, the successfully packaged CV-A16-CXCL13 recombinant virus was obtained. Expression of CXCL13 and structural protein VP1 were detected at the gene level by PCR with virus-infected cells. Immunoblotting and immunofluorescence results showed that CXCL13 and VP1 protein were simultaneously expressed in infected cells. Electron microscopy showed that the recombinant virus particles were 20～30 nm in diameter. The proliferation curve showed that the recombinant virus was able to replicate normally in the cell. This paper successfully constructed the CV-A16-CXCL13 virus, which will be useful for the study of the characteristics of CV-A16 infection and development of vaccines.
引文
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[14]Crotty S.T follicular helper cell differentiation,func-tion,and roles in disease[J].Immunity,2014,41(4):529-542.
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[17]Liu L,Mo Z,Liang Z,Zhang Y,Li R,Ong K C,Wong K T,Yang E,Che Y,Wang J,Dong C,Feng M,Pu J,Wang L,Liao Y,Jiang L,Tan S H,David P,Huang T,Zhou Z,Wang X,Xia J,Guo L,Wang L,Xie Z,Cui W,Mao Q,Liang Y,Zhao H,Na R,Cui P,Shi H,Wang J,Li Q.Immunity and clinical effi-cacy of an inactivated enterovirus 71 vaccine in healthy Chinese children:a report of further observations[J].BMC Med,2015,13:226.
[18]Wang J,Qi S,Zhang X,Zhang Y,Liu L,Che Y,He Z,Zhao Y,Lu S,Yu W,Li Q.Coxsackievirus A 16 in-fection does not interfere with the specific immune re-sponse induced by an enterovirus 71 inactivated vaccine in rhesus monkeys[J].Vaccine,2014,32(35):4436-4442.
[19]Takahashi S,Liao Q,Van Boeckel T P,Xing W,Sun J,Hsiao V Y,Metcalf C J,Chang Z,Liu F,Zhang J,Wu J T,Cowling B J,Leung G M,Farrar J J,Van Doorn H R,Grenfell B T,Yu H.Hand,foot,and mouth disease in China:modeling epidemic dynamics of enterovirus serotypes and implications for vaccination[J/OL].PLoS Med,2016,13(2):e1001958.
[20]Qi An W,Guo Su Z,Wen Pan R,Ping Yang B,Chao Zhang Y,Shi L,Li Q.The immunogenicity and protec-tion effect of the BPL-inactivated CV-A16 vaccine in dif-ferent animal systems[J].Hum Vaccin Immunother,2014,10(3):628-639.
[21]Ku Z,Liu Q,Ye X,Cai Y,Wang X,Shi J,Li D,Jin X,An W,Huang Z.A virus-like particle based bivalent vaccine confers dual protection against enterovirus 71and coxsackievirus A16 infections in mice[J].Vaccine,2014,32(34):4296-4303.
[22]Wang J,Zhang Y,Zhang X,Hu Y,Dong C,Liu L,Yang E,Che Y,Pu J,Wang X,Song J,Liao Y,Feng M,Liang Y,Zhao T,Jiang L,He Z,Lu S,Wang L,Li Y,Fan S,Guo L,Li Q.Pathologic and immunolog-ic characteristics of coxsackievirus A16 infection in rhe-sus macaques[J].Virology,2017,500:198-208.
[23]Song J,Hu Y,Hu Y,Wang J,Zhang X,Wang L,Guo L,Wang Y,Ning R,Liao Y,Zhang Y,Zheng H,Shi H,He Z,Li Q,Liu L.Global gene expression anal-ysis of peripheral blood mononuclear cells in rhesus mon-key infants with CV-A16 infection-induced HFMD[J].Virus Res,2016,214:1-10.
[24]Wang J,Pu J,Liu L,Che Y,Liao Y,Wang L,Guo L,Feng M,Liang Y,Fan S,Cai L,Zhang Y,Li Q.Clinical and Associated Immunological Manifestations of HFMD Caused by Different Viral Infections in Children[J].Glob Pediatr Health,2016,3:2333794x16643723.
[25]Havenar-Daughton C,Lindqvist M,Heit A,Wu J E,Reiss S M,Kendric K,Bélanger S,Kasturi S P,Landa-is E,Akondy R S,Mcguire H M,Bothwell M,Vagefi P A,Scully E,Tomaras G D,Davis M M,Poignard P,Ahmed R,Walker B D,Pulendran B,Mcelrath MJ,Kaufmann D E,Crotty S.CXCL13 is a plasma bio-marker of germinal center activity[J].Proc Natl Acad Sci U S A,2016,113(10):2702-2707.
[2]Ren J,Wang X,Zhu L,Hu Z,Gao Q,Yang P,Li X,Wang J,Shen X,Fry E E,Rao Z,Stuart D I.Struc-tures of coxsackievirus A16 capsids with native antigenic-ity:Implications for particle expansion,receptor bind-ing,and immunogenicity[J].J Virol,2015,89(20):10500-10511.
[3]Ang L W,Tay J,Phoon M C,Hsu J P,Cutter J,James L,Goh K T,Chow V T.Seroepidemiology of coxsackievirus A6,coxsackievirus A16,and enterovirus71 infections among children and adolescents in Singa-pore,2008-2010[J/OL].PLoS One,2015,10(5):e0127999.
[4]Rao D C,Naidu J R,Maiya P P,Babu A,Bailly J L.Large-scale HFMD epidemics caused by Coxsackievirus A16 in Bangalore,India during 2013 and 2015[J].In-fect Genet Evol,2017,55:228-235.
[5]孙立梅.广东省手足口病感染柯萨奇病毒A组16型病例流行特征分析;2017新发传染病研究热点研讨会,中国广东广州,F,2017[C].
[6]Yan X F,Gao S,Xia J F,Ye R,Yu H,Long J E.Epi-demic characteristics of hand,foot,and mouth disease in Shanghai from 2009 to 2010:Enterovirus 71 subgeno-type C4 as the primary causative agent and a high inci-dence of mixed infections with coxsackievirus A16[J].Scand J Infect Dis,2012,44(4):297-305.
[7]Zhang Y,Zhu Z,Yang W,Ren J,Tan X,Wang Y,Mao N,Xu S,Zhu S,Cui A,Zhang Y,Yan D,Li Q,Dong X,Zhang J,Zhao Y,Wan J,Feng Z,Sun J,Wang S,Li D,Xu W.An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand foot and mouth disease in Fuyang city of China[J].Vi-rol J,2010,7:94-94.
[8]Liu W,Wu S,Xiong Y,Li T,Wen Z,Yan M,Qin K,Liu Y,Wu J.Co-circulation and genomic recombina-tion of coxsackievirus A16 and enterovirus 71 during a large outbreak of hand,foot,and mouth disease in Cen-tral China[J/OL].PLoS One,2014,9(4):e96051.
[9]Wang Z,Li M,Zhou M,Zhang Y,Yang J,Cao Y,Wang K,Cui M,Chen H,Fu Z F,Zhao L,Williams B R G.A novel rabies vaccine expressing CXCL13 en-hances humoral immunity by recruiting both T follicular helper and germinal center B cells[J/OL].J Virol,2017,91(3).pii:e01956-16.
[10]Legler D F,Loetscher M,Roos R S,Clark-Lewis I,Baggiolini M,Moser B.B cell-attracting chemokine 1,a human CXC chemokine expressed in lymphoid tissues,selectively attracts B lymphocytes via BLR1/CXCR5[J].J Exp Med,1998,187(4):655-660.
[11]Ansel K M,Harris R B S,Cyster J G.CXCL13 is Re-quired for B1 cell homing,natural antibody production,and body cavity immunity[J].Immunity,2002,16(1):67-76.
[12]Ansel K M,Ngo V N,Hyman P L,Luther S A,F?rster R,Sedgwick J D,Browning J L,Lipp M,Cys-ter J G.A chemokine-driven positive feedback loop orga-nizes lymphoid follicles[J].Nature,2000,406(6793):309-314.
[13]Zhang Y,Garcia-Ibanez L,Toellner K M.Regulation of germinal center B-cell differentiation[J].Immunol Rev,2016,270(1):8-19.
[14]Crotty S.T follicular helper cell differentiation,func-tion,and roles in disease[J].Immunity,2014,41(4):529-542.
[15]Ma C S,Deenick E K,Batten M,Tangye S G.The ori-gins,function,and regulation of T follicular helper cells[J].J Exp Med,2012,209(7):1241-1253.
[16]Gong M,Zhu H,Zhou J,Yang C,Feng J,Huang X,Ji G,Xu H,Zhu P.Cryo-electron microscopy study of insect cell-expressed enterovirus 71 and coxsackievirus A16 virus-like particles provides a structural basis for vaccine development[J].J Virol,2014,88(11):6444-6452.
[17]Liu L,Mo Z,Liang Z,Zhang Y,Li R,Ong K C,Wong K T,Yang E,Che Y,Wang J,Dong C,Feng M,Pu J,Wang L,Liao Y,Jiang L,Tan S H,David P,Huang T,Zhou Z,Wang X,Xia J,Guo L,Wang L,Xie Z,Cui W,Mao Q,Liang Y,Zhao H,Na R,Cui P,Shi H,Wang J,Li Q.Immunity and clinical effi-cacy of an inactivated enterovirus 71 vaccine in healthy Chinese children:a report of further observations[J].BMC Med,2015,13:226.
[18]Wang J,Qi S,Zhang X,Zhang Y,Liu L,Che Y,He Z,Zhao Y,Lu S,Yu W,Li Q.Coxsackievirus A 16 in-fection does not interfere with the specific immune re-sponse induced by an enterovirus 71 inactivated vaccine in rhesus monkeys[J].Vaccine,2014,32(35):4436-4442.
[19]Takahashi S,Liao Q,Van Boeckel T P,Xing W,Sun J,Hsiao V Y,Metcalf C J,Chang Z,Liu F,Zhang J,Wu J T,Cowling B J,Leung G M,Farrar J J,Van Doorn H R,Grenfell B T,Yu H.Hand,foot,and mouth disease in China:modeling epidemic dynamics of enterovirus serotypes and implications for vaccination[J/OL].PLoS Med,2016,13(2):e1001958.
[20]Qi An W,Guo Su Z,Wen Pan R,Ping Yang B,Chao Zhang Y,Shi L,Li Q.The immunogenicity and protec-tion effect of the BPL-inactivated CV-A16 vaccine in dif-ferent animal systems[J].Hum Vaccin Immunother,2014,10(3):628-639.
[21]Ku Z,Liu Q,Ye X,Cai Y,Wang X,Shi J,Li D,Jin X,An W,Huang Z.A virus-like particle based bivalent vaccine confers dual protection against enterovirus 71and coxsackievirus A16 infections in mice[J].Vaccine,2014,32(34):4296-4303.
[22]Wang J,Zhang Y,Zhang X,Hu Y,Dong C,Liu L,Yang E,Che Y,Pu J,Wang X,Song J,Liao Y,Feng M,Liang Y,Zhao T,Jiang L,He Z,Lu S,Wang L,Li Y,Fan S,Guo L,Li Q.Pathologic and immunolog-ic characteristics of coxsackievirus A16 infection in rhe-sus macaques[J].Virology,2017,500:198-208.
[23]Song J,Hu Y,Hu Y,Wang J,Zhang X,Wang L,Guo L,Wang Y,Ning R,Liao Y,Zhang Y,Zheng H,Shi H,He Z,Li Q,Liu L.Global gene expression anal-ysis of peripheral blood mononuclear cells in rhesus mon-key infants with CV-A16 infection-induced HFMD[J].Virus Res,2016,214:1-10.
[24]Wang J,Pu J,Liu L,Che Y,Liao Y,Wang L,Guo L,Feng M,Liang Y,Fan S,Cai L,Zhang Y,Li Q.Clinical and Associated Immunological Manifestations of HFMD Caused by Different Viral Infections in Children[J].Glob Pediatr Health,2016,3:2333794x16643723.
[25]Havenar-Daughton C,Lindqvist M,Heit A,Wu J E,Reiss S M,Kendric K,Bélanger S,Kasturi S P,Landa-is E,Akondy R S,Mcguire H M,Bothwell M,Vagefi P A,Scully E,Tomaras G D,Davis M M,Poignard P,Ahmed R,Walker B D,Pulendran B,Mcelrath MJ,Kaufmann D E,Crotty S.CXCL13 is a plasma bio-marker of germinal center activity[J].Proc Natl Acad Sci U S A,2016,113(10):2702-2707.