摘要
猪血凝性脑脊髓炎病毒(porcine hemagglutinating encephalomyelitis virus，PHEV)是引起猪血凝性脑脊髓炎(porcine hemagglutinating encephalomyelitis)的致病原，属于β冠状病毒属的成员。PHEV的主要感染对象为哺乳期的仔猪。一旦病毒感染仔猪，其传播速度很快，且会造成很高的死亡率。PHEV与其它冠状病毒结构相似，包含了五种结构蛋白，即小膜蛋白(E蛋白)、膜蛋白(M蛋白)、刺突蛋白(S蛋白)、核衣壳蛋白(N蛋白)以及血凝素——酯酶蛋白(HE蛋白)。
通过对其它冠状病毒的研究发现，S蛋白在病毒入侵和感染细胞过程中，起到了关键的作用。冠状病毒S蛋白分为S1和S2两个功能域，这两个功能域在S蛋白中发挥着各自的作用，其中S1功能域的主要功能是能够与易感细胞表面的受体结合，并能够诱导S2功能域与细胞膜的融合。S1功能域中包含着与细胞受体相结合的区域，即受体结合域(RBD)。而对于RBD的研究，则有助于更深层次的了解病毒入侵机制以及研发出更有针对性的病毒疫苗。
本研究依据冠状病毒S蛋白划分标准，通过与部分冠状病毒S蛋白氨基酸序列进行比对，将PHEV S蛋白划分为S1和S2功能域，即第1-794位氨基酸被定义为是S1功能域，第795-1349位氨基酸被定义为S2功能域。将PHEV与其它冠状病毒的理化性质进行比较分析，以及对S蛋白亲水性参数，可及性参数，β-转角等进行预测后，证明了S蛋白具有较强的亲水能力，在S1功能域上存在着10个能够与受体发生结合的可及性片段。
参照S蛋白的生物信息学分析结果，将S蛋白S1功能域分为三段基因片段，分别命名为：S1-291、S277-794和S548-868，把三段基因片段与原核表达载体pGEX-4T-1的线性化片段连接，构建重组表达载体pGEX-4T-1-S1-291，pGEX-4T-1-S548-868和pGEX-4T-1-S277-794，将重组表达载体分别转化入BL21感受态细胞中，经IPTG诱导，三段蛋白成功的以可溶性表达。在本课题组的前期工作中，已经发现NCAM能够与PHEV S蛋白S1功能域发生相互作用。并且，通过实验证实了NCAM参与了猪血型性脑脊髓病毒侵入神经细胞的过程，在PHEV侵染神经细胞过程中发挥着作用，其极有可能为PHEV在N2a细胞上的受体。因此，本研究将NCAM基因片段连接到pET28a表达载体，构建重组表达载体pET-NC，转化入BL21感受态细胞中，经IPTG诱导在大肠杆菌中获得了可溶性表达蛋白。
为了找寻PHEV S1功能域上能够与NCAM蛋白发生相互作用的区域，本研究使用GST pull-down实验来筛选相互作用的片段，结果发现S277-794与NCAM存在着相互作用，而其它两段蛋白（S1-291和S548-868）没有这种结合能力。随后使用了酵母双杂交实验对结果进行验证，结果证实了S277-794能够与NCAM发生相互作用，而这种相互作用不存在于S1-291与NCAM以及S548-868与NCAM之间。然而，在S277-794与S1-291之间存在着相互重叠的部分，并且S277-794与S548-868之间也有相互重叠的部分。因此，依据这些结果，本研究预测出一段包含有258aa的小片段(S291-548)，这个小片段很有可能为PHEV S蛋白上介导与NCAM发生相互作用的关键区域，其很有可能为PHEV S蛋白上的RBD。
为了对S291-548进行进一步的分析，本研究将S291-548进行大肠杆菌体外表达，获得了含有目的蛋白片段的融合蛋白。将S291-548与NCAM作用后的混合液与吸附在Glutathione-Sepharose4B颗粒上的S277-794-GST融合蛋白进行反应，使用抑制蛋白相互作用的GST pull-down实验来证明S291-548能够与NCAM的相互作用，结果表明，S291-548重组蛋白因为与NCAM发生了结合而抑制了S277-794-GST与NCAM的结合，证明了筛选出的S291-548能够与NCAM发生相互作用。
同时，使用S291-548的重组表达蛋白免疫家兔，制备多克隆抗体，将多抗与病毒等体积混合后，接种到铺满单层的N2a细胞中，接毒48h后，分别将阴性对照组、实验组和阳性对照组的细胞培养液上清取出进行血凝实验，结果表明实验组HEV血凝价为1:22，而阳性对照组血凝价为1:24，两组间血凝价有明显差异。证明S291-548能够抑制PHEV对N2a细胞的感染。这些结果说明S291-548可能是NCAM的一个潜在的RBD，这一结果的获得对于进一步分析PHEV S蛋白的结构与功能以及设计更具有靶向性的疫苗奠定了基础。
Porcine hemagglutinating encephalomyelitis coronavirus (PHEV) is a member ofthe Coronaviridae family, which causes porcine encephalomyelitis. PHEVpredominantly affects piglets. When PHEV infects the piglets, its transmission speedwill be quick and with a high mortality rate. The major structural proteins of the virusnamed small membrance(E), membrance(M),spike(S),nucleocapsid (N) protein andhemagglutinin-esterase (HE).
Base on the study of other coronavirus, the Coronavirus Spike (S) protein isdivided into S1functional domain and S2functional domain.The S1functionaldomain initiates entry of viruses into cells by binding to cell surface receptors, and theS2functional domain helps viral fusion with cellular membranes. S1function domaincontains the receptor binding domain (RBD). Given this, it is necessary that screeningand identification of the RBD to provide some valuable information for developmentof diagnosis technology and novel vaccine of PHEV.
According to the presence of conserved nonamer and the GxCx motifs at theproteolytic cleavage site of S protein in other members of coronavirus, PHEV Sprotein was divided into S1and S2function domain, The1-794aa of PHEV S proteinis the S1and the795-1349aa is the S2.Therefore, based on standard of coronavirus Sprotein, and compared with some other coronavirus S protein amino acid sequence, Sprotein is with strong hydrophilic ability, and there are10domains maybe combinewith the receptor accessibility on the S1.
Considering to accessibility domains predicted, the S1functional domains ofPHEV S protein was divided into three truncated fusion proteins, S1-291、S277-794andS548-868.The plasmids pGEXS1-291, pGEXS277-794, pGEXS548-868were individuallytransformed in E. coli BL21cells separately and three truncated fusion proteins wereexpressed successfully.In the preliminary work of our project group, it has found thatthe neural cell adhesion molecule (NCAM) interacts with S1functional domain of PHEV S protein.And it was confirmed that NCAM participates in the process bywhich PHEV infects neurons.NACM may be a receptor for PHEV in N2acells.Therefore, pET-NC was transformed in E. coli BL21cells and the NCAM genewas expressed successfully,too.
To screened the domains that could interact with NCAM,this study used GSTpull down and the interaction was further confirmed by a yeast wo-hybrid systemassay. The results showed that S277-794were identified to interact with NCAM,whileS1-291and S548-868not.S277-794is located in S1subunit. In addition,because S1-291orS548-868could not interact with NCAM, and there are overlapping region betweenS277-794and S1-291as well as S277-794and S548-868, we conclude that the S1subunit maycontain a minimal cellular receptor binding region. These findings suggested that the258-amino-acid fragment, residues291to548, may be the NCAM RBD of the Sprotein.Finally, a small fragment (258-amino-acid fragment, residues291to548) onthe PHEV S protein was posited to be the minimum number of amino acids necessaryto interact with NCAM, and it is very likely to the RBD that mediates PHEV bindingto NCAM.
In order to further analysis for fragment, His fusion gene of the S291-548wasexpressed in BL21. The mixture contained S291-548and NCAM was incubated withS277-794-GST immobilized Glutathione Sepharose4B.The inhibiting protein interactionGST pull-down experiment shows that S291-548completely blocked the bindingbetween S277-794and NCAM，further proved that S291-548is able to interact with theNCAM.
The polyclonal antibody of S291-548was prepared with immunized rabbit. Aftermixing the prepared polyclonal antibody and PHEV, and then was inoculated to theneural cells with the HEV. At48h after the inoculation, the hemagglutination titer ofthe HEV in the supernatant fluid of the cell culture was1:22test group,and thehemagglutination titer of HEV in the supernatant fluid of the cell culture was1:24inthe control group. A difference of two gradients existed between the hemagglutinationtiters of the two groups, thus the difference was significant.Results showed thatS291-548protein antibodies could inhibit the proliferation of PHEV in the cell. Theseresults provided evidences that S291-548may be a potential of NCAM receptor binding domain. Meanwhile,these data could provide the basis for the development ofidentifying the effective target epitope for a S protein-based vaccine and furtherstructure and function analysis of S protein.
引文
[1] Cavanagh, D. Nidovirales: a new order comprising Coronaviridae andArteriviridae[J]. Arch Virol,1997,142(3):629-33.
[2] Lai, M. M., and D. Cavanagh. The molecular biology of coronaviruses[J]. AdvVirus.Res,1997,48:1-100.
[3] Boursnell, M. E., Brown, T. D., Foulds, I. J., Green, P. F., Tomley, F. M., andBinns, M M.Completion of the sequence of the genome of the coronavirus avianinfectious bronchitis virus[J]. J Gen Virol,1987,68:57-77.
[4] Eleouet, J. F., Rasschaert, D., Lambert, P., Levy, L., Vende, P., and Laude,H.Complete sequence (20kilobases) of the polyprotein-encoding gene1oftransmissible gastroenteritis virus[J]. Virology,1995,206(2):817-22.
[5] Herold, J., Raabe, T., Schelle-Prinz, B., and Siddell, S. G. Nucleotide sequence ofthe human coronavirus229E RNA polymerase locus[J]. Virology,1993,195(2):680-91.
[6] Lai, M. M., and Cavanagh, D. The molecular biology of coronaviruses[J]. AdvVirus Res,1997,48:1-100.
[7] Lee，H. J., Shieh, C. K., Gorbalenya, A. E., Koonin, E. V., La Monica, N., Tuler, JBagdzhadzhyan, A., and Lai, M. M. The complete sequence (22kilobases) ofmurine coronavirus gene1encoding the putative proteases and RNApolymerase[J]. Virology,1991,180(2):567-82.
[8] Anand, Kanchan, et al."Structure of coronavirus main proteinase revealscombination of a chymotrypsin fold with an extra α‐helical domain."TheEMBO journal21.13(2002):3213-3224.
[9] Lewicki, Daniel N., and Thomas M. Gallagher."Quaternary structure ofcoronavirus spikes in complex with carcinoembryonic antigen-related celladhesion molecule cellular receptors." Journal of Biological Chemistry277.22(2002):19727-19734.
[10] Anand, Kanchan, et al."Coronavirus main proteinase (3CLpro) structure: basisfor design of anti-SARS drugs." Science300.5626(2003):1763-1767.
[11] Lai, Michael MC, and David Cavanagh."The molecular biology ofcoronaviruses." Advances in virus research48.48(1997):1-100.
[12] Anand, Kanchan, et al."Structure of coronavirus main proteinase revealscombination of a chymotrypsin fold with an extra α‐helical domain."TheEMBO journal21.13(2002):3213-3224.
[13] Lewicki, Daniel N., and Thomas M. Gallagher."Quaternary structure ofcoronavirus spikes in complex with carcinoembryonic antigen-related celladhesion molecule cellular receptors." Journal of Biological Chemistry277.22(2002):19727-19734.
[14]Ziebuhr, John."Molecular biology of severe acute respiratory syndromecoronavirus." Current opinion in microbiology7.4(2004):412-419.
[15] Bosch, Berend Jan, et al."The coronavirus spike protein is a class I virus fusionprotein: structural and functional characterization of the fusion corecomplex."Journal of virology77.16(2003):8801-8811.
[16] Spiga, Ottavia, et al."Molecular modelling of S1and S2subunits of SARScoronavirus spike glycoprotein." Biochemical and biophysical researchcommunications310.1(2003):78-83.
[17] Sturman, Lawrence S., K. V. Holmes, and J. Behnke."Isolation of coronavirusenvelope glycoproteins and interaction with the viral nucleocapsid." Journal ofvirology33.1(1980):449-462.
[18] Sturman, Lawrence S., and Kathryn V. Holmes."The molecular biology ofcoronaviruses." Adv. Virus Res28(1983):35-112.
[19] Li, Fang, et al."Structure of SARS coronavirus spike receptor-binding domaincomplexed with receptor." Science309.5742(2005):1864-1868.
[20] Zeng, Qinghong, et al."Structure of coronavirus hemagglutinin-esterase offersinsight into corona and influenza virus evolution." Proceedings of the NationalAcademy of Sciences105.26(2008):9065-9069.
[21] Graham, Rachel L., Eric F. Donaldson, and Ralph S. Baric."A decade afterSARS: strategies for controlling emerging coronaviruses." Nature ReviewsMicrobiology11.12(2013):836-848.
[22] Holmes, K. V. Coronaviruses, p.1187-1203. In e. a. D. Knipe (ed.), Fields'Virology,4th ed,vol.1. Lippincott Williams and Wilkins, Philadelphia.2001.
[23] Lai, M. M. C., and K. V. Holmes. Coronaviridae: The Viruses and theirReplication, p.1163-1185. In D. Knipe, et al.(ed.), Fields' Virology,4ed, vol.1.Lippincott-Williams and Wilkins, Philadelphia.2001.
[24] Machamer, C. E., Grim, M. G., Esquela, A., Chung, S. W., Rolls, M., Ryan, K.,and Swift, A.M. Retention of a cis Golgi protein requires polar residues on oneface of a predicted alpha-helix in the transmembrane domain[J]. Mol Biol Cell,1993,4(7):695-704.
[25] Yamanaka M, Crisp T, Brown R, Dale B. Nucleotide sequence of theinter-structural gene region of feline infectious peritonitis virus[J]. Virus Genes.1998;16(3):317-8.
[26] Antoine A F de Vries, Marian C Horzinek, Peter J M Rottier, Raoul J de Groot.The genome organization of the nidovirales: similarities and differences betweenarteri-, toro-, and coronaviruses[J]. Virology,1997.8:33-47.
[27] Brian DA, Baric RS. Coronavirus genome structure and replication[J]. Curr TopMicrobiol Immunol.2005;287:1-30.
[28] Masters, Paul S."The molecular biology of coronaviruses." Advances in virusresearch66(2006):193-292.
[29] Vennema H, Rossen JW, Wesseling J, Horzinek MC, Rottier PJ. Genomicorganization and expression of the3’ end of the canine and feline entericcoronaviruses[J]. Virology.1992,191(1):134-140.
[30] Xu, Yanhui, et al."Crystal structure of severe acute respiratory syndromecoronavirus spike protein fusion core." Journal of Biological Chemistry279.47(2004):49414-49419.
[31] Popova R, Zhang X. The spike but not the hemagglutinin/esterase protein ofbovine coronavims is necessary and sufficient for viral infection[J]. Virology.2002.294:222-236.
[32]Sturman, Lawrence S., and Kathryn V. Holmes."The molecular biology ofcoronaviruses." Adv. Virus Res28(1983):35-112.
[33]Li, Fang, et al."Structure of SARS coronavirus spike receptor-binding domaincomplexed with receptor." Science309.5742(2005):1864-1868.
[34] Zeng, Qinghong, et al."Structure of coronavirus hemagglutinin-esterase offersinsight into corona and influenza virus evolution." Proceedings of the NationalAcademy of Sciences105.26(2008):9065-9069.
[35] Masters, Paul S."The molecular biology of coronaviruses." Advances in virusresearch66(2006):193-292.
[36] Gallagher T M, Buchmeier M J. Coronavirus spike proteins in viral entry andpathogenesis[J]. Virology,2001.279:371-374.
[37]Xu, Yanhui, et al."Crystal structure of severe acute respiratory syndromecoronavirus spike protein fusion core." Journal of Biological Chemistry279.47(2004):49414-49419.
[38]Tan, Kemin, et al."Crystal structure of murine sCEACAM1a [1,4]: a coronavirusreceptor in the CEA family." The EMBO journal21.9(2002):2076-2086.
[39]Spaan, W., D. Cavanagh, and M. C. Horzinek."Coronaviruses: structure andgenome expression." J. gen. Virol69.Pt12(1988):2939-2952.
[40]Fraser, R. D., et al."Molecular structure of the cell-attachment protein of reovirus:correlation of computer-processed electron micrographs with sequence-basedpredictions." Journal of virology64.6(1990):2990-3000.
[41]赵荣乐,郑光宇.冠状病毒研究进展[J].生物学通报,2003,38(6):3-5.
[42] Sturman, Lawrence S."Characterization of a coronavirus: I. Structural proteins:Effects of preparative conditions on the migration of protein in polyacrylamidegels." Virology77.2(1977):637-649.
[43] King, Bernadette, and David A. Brian."Bovine coronavirus structuralproteins."Journal of virology42.2(1982):700-707.
[44] Saikatendu, Kumar Singh, et al."Structural basis of severe acute respiratorysyndrome coronavirus ADP-ribose-1″-phosphate dephosphorylation by aconserved domain of nsP3." Structure13.11(2005):1665-1675.
[45] Ziebuhr J. The coronavirus replicase[J]. Curr Top Microbiol Immunol.2005;287:57-94.
[46] Sawicki S G, Sawicki D L. A new model for coronavirus transcription.[J] AdvExp Med Biol,1998.440:215-21915
[47] Cavanagh, David."Coronavirus IBV: structural characterization of the spikeprotein." Journal of general virology64.12(1983):2577-2583.
[48] Tripet, Brian, et al."Structural characterization of the SARS-coronavirus spike Sfusion protein core." Journal of Biological Chemistry279.20(2004):20836-20849.
[49] Lee, Vannajan Sanghiran, et al."Structure and dynamics of SARS coronavirusproteinase: The primary key to the designing and screening for anti-SARSdrugs." Science Asia29(2003):181-188.
[50] Milleri W Alien, Gennadiy Koev. Minireview: synthesis of subgenomic RNAs bypositive-strand RNA viruses[J]. Virology,2000.273,1-8.
[51] Sit T L, Vaewhongs A A, Lommel S A. RNA-mediated transactivation oftranscription from a viral RNA[J]. Science,1998.281:829-832.
[52] Sawicki D, Wang T, Sawicki S. The RNA structures engaged in replication andtranscription of the A59strain of mouse hepatitis virus[J]. J Gen Virol.2001Feb;82(2):385-96.
[53] Spiga, Ottavia, et al."Molecular modelling of S1and S2subunits of SARScoronavirus spike glycoprotein." Biochemical and biophysical researchcommunications310.1(2003):78-83.
[54] Xu, Yanhui, et al."Structural Basis for Coronavirus-mediated Membrane FusionCRYSTAL STRUCTURE OF MOUSE HEPATITIS VIRUS SPIKE PROTEINFUSION CORE." Journal of Biological Chemistry279.29(2004):30514-30522.
[55] De Groot, R. J., et al."Evidence for a coiled-coil structure in the spike proteins ofcoronaviruses." Journal of molecular biology196.4(1987):963-966.
[56] Wang Y, Zhang X. The leader RNA of coronavirus mouse hepatitis virus containsan enhancer-like element for subgenomic mRNA transcription[J]. J Virol.2000Nov;74(22):10571-80.
[57] Kuo L, Masters PS. Genetic evidence for a structural interaction between thecarboxy termini of the membrane and nucleocapsid proteins of mouse hepatitisvirus[J].J Virol.2002May;76(10):4987-99.
[58] Krishna Narayanan, Akihiko Maeda, Junko Maeda, Shinji Makino.Characterization of the coronavirus M protein and nucleocapsid interaction ininfected cells[J]. J Virol,2000.74(17):8127-8134.
[59] Supekar, Vinit M., et al."Structure of a proteolytically resistant core from thesevere acute respiratory syndrome coronavirus S2fusion protein." Proceedingsof the National Academy of Sciences101.52(2004):17958-17963.
[60] Hogue, Brenda G., and David A. Brian."Structural proteins of human respiratorycoronavirus OC43." Virus research5.2(1986):131-144.
[61] Sun, Haifang, et al."Molecular cloning, expression, purification, and massspectrometric characterization of3C-like protease of SARS coronavirus."Proteinexpression and purification32.2(2003):302-308.
[62] Du, Qishi, et al."Molecular modeling and chemical modification for findingpeptide inhibitor against severe acute respiratory syndrome coronavirus mainproteinase." Analytical biochemistry337.2(2005):262-270.
[63] Hofmann, MARTIN A., PHIROZE B. Sethna, and David A. Brian."Bovinecoronavirus mRNA replication continues throughout persistent infection in cellculture." Journal of virology64.9(1990):4108-4114.
[64] Risco, Cristina, et al."The transmissible gastroenteritis coronavirus contains aspherical core shell consisting of M and N proteins." Journal of virology70.7(1996):4773-4777.
[65] Blanchard, Jan E., et al."High-throughput screening identifies inhibitors of theSARS coronavirus main proteinase." Chemistry&biology11.10(2004):1445-1453.
[66] Liu, D. X., and Inglis, S. C. Association of the infectious bronchitis virus3cprotein with the virion envelope[J]. Virology,1991,185(2):911-7.
[67] Lim KP, Liu DX. The missing link in coronavirus assembly. Retention of theavian coronavirus infectious bronchitis virus envelope protein in the pre-Golgicompartments and physical interaction between the envelope and membraneproteins[J]. J Biol Chem.2001May18;276(20):17515-23.
[68] Dede Haan CA, Smeets M, Vernooij F, Vennema H, Rottier PJ. Mapping of thecoronavirus membrane protein domains involved in interaction with the spikeprotein[J]. J Virol.1999Sep;73(9):7441-52.
[69] Huang, Y., Yang, Z. Y., Kong, W. P., and Nabel, G. J. Generation of syntheticsevere acute respiratory syndrome coronavirus pseudoparticles: implications forassembly and vaccine production[J]. J Virol,2004,78(22):12557-65.
[70] Enjuanes L. Membrane protein molecules of transmissible gastroenteritiscoronavirus also expose the carboxy-terminal region on the external surface ofthe virion[J]. J Virol.1995Sep;69(9):5269-5277.
[71] Pachuk, Catherine J., et al."Molecular cloning of the gene encoding the putativepolymerase of mouse hepatitis coronavirus, strain A59." Virology171.1(1989):141-148.
[72] Escors D, Ortego J, Laude H, Enjuanes L.The membrane M protein carboxyterminus binds to transmissible gastroenteritis coronavirus core and contributesto core stability[J]. J Virol.2001Feb;75(3):1312-24.
[73] Siddell, Stuart, Helmut Wege, and Volker Ter Meulen."The biology ofcoronaviruses." Journal of General Virology64.4(1983):761-776.
[74] Xu, Xiang, et al."Molecular model of SARS coronavirus polymerase:implications for biochemical functions and drug design." Nucleic acidsresearch31.24(2003):7117-7130.
[75] Schultze B, Enjuanes L, Cavanagh D, Herrler G N-acetylneuraminic acid plays acritical role for the haemagglutinating activity of avian infectious bronchitis virusand porcine transmissible gastroenteritis virus[J]. Adv Exp Med Biol,1993.342:305-310.
[76] Parker, Monica M., and Paul S. Masters."Sequence comparison of the N genesof five strains of the coronavirus mouse hepatitis virus suggests a three domainstructure for the nucleocapsid protein." Virology179.1(1990):463-468.
[77] Kim L, Hayes J, Parwani P, et al. Molecular Characterization and Pathogenesis ofTransmissible Gastroenteritis Virus (TGEV) and Respirartory Coronavirus(PRCV) Field Isolates Co-circulating In a Swine Herd.[J] Arch Virology.2000,145:1133-1147.
[78] Compton, S. R.Rogers, D. B. Holmes, K. V. Fertsch, D.Remenick, J.andMcGowan, J. J.In vitro replication of mouse hepatitis virus strain A59[J]. J Virol,1987,61(6):1814-20.
[79] Supekar, Vinit M., et al."Structure of a proteolytically resistant core from thesevere acute respiratory syndrome coronavirus S2fusion protein." Proceedingsof the National Academy of Sciences101.52(2004):17958-17963.
[80] Ratia, Kiira, et al."Severe acute respiratory syndrome coronavirus papain-likeprotease: structure of a viral deubiquitinating enzyme." Proceedings of theNational Academy of Sciences103.15(2006):5717-5722.
[81] Salanueva, I igo J., José L. Carrascosa, and Cristina Risco."Structuralmaturation of the transmissible gastroenteritis coronavirus." Journal ofvirology73.10(1999):7952-7964.
[82] Lai, M. M. C. and K. V. Holmes. Coronaviridae: The Viruses and theirReplication, p.1163-1185. In D. Knipe, et al.(ed.), Fields' Virology,4ed, vol.1.Lippincott-Williams and Wilkins, Philadelphia.2001.
[83] S. Becker, P. Bates, H. Hofmann, and S. Pohlmann. DC-SIGN and DC-SIGNR Interact with the glycoprotein of Marburg virus and the S protein of severe acuterespiratory syndrome coronavirus[J]. J Virol,2004,78:12090-12095.
[84]殷震,刘景华.动物病毒学[M].北京:科学出版社,1997,690-692.
[85] Vennema, H., A. Poland, J. Foley, and N. C. Pedersen. Feline infectiousperitonitis viruses arise by mutation from endemic feline entericcoronaviruses[J].Virology,1998,243:150-157.
[86] Gorbalenya, A. E., E. J. Snijder, and W. J. Spaan. Severe acute respiratorysyndrome coronavirus phylogeny: toward consensus[J]. J Virol,2004,78:7863-7866.
[87] Lau, S. K., P. C. Woo, K. S. Li, Y. Huang, H. W. Tsoi, B. H. Wong, S. S. Wong, S.Y. Leung,K. H. Chan, and K. Y. Yuen. Severe acute respiratory syndromecoronavirus-like virus in Chinese horseshoe bats[J]. Proc Natl Acad Sci U S A,2005,102:14040-14045.
[88] Saikatendu, Kumar Singh, et al."Ribonucleocapsid formation of severe acuterespiratory syndrome coronavirus through molecular action of the N-terminaldomain of N protein." Journal of virology81.8(2007):3913-3921.
[89] Drosten, C., Gunther, S., Preiser, W., van der Werf, S., Brodt, H. R., Becker, S.,Rabenau, H,Panning, M., Kolesnikova, L., Fouchier, R. A., Bergen A., Burguiere,A. M., Cinatl, J,Eickmann, M, Escriou, N, Grywna, K, Kramme, S Manuguerra,J. C, Muller, S., Rickerts,V, Sturmer, M., Vieth, S., Klenk, H. D., Osterhaus, A. D.Schmitz, H., and Doerr, H. W.Identification of a novel coronavirus in patientswith severe acute respiratory syndrome[J]. N Engl J Med,2003,348(20):1967-76.
[90] 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, andAnderson, L. J. A novel coronavirus associated with severe acute respiratorysyndrome[J]. NEngl Jmed,2003,348(20):1953-66.
[91] Collins A R. Identification of120kD and30kD receptors for human coronavirusOC43in cell membrane preparations from newborn mouse brain [J]. Adv ExpMed Biol,1995,380(387-390.
[92] Matsuyama S, Taguchi F. Receptor-induced conformational changes of murinecoronavirus spike protein [J]. J Virol,2002,76(23):11819-11826.
[93] Zelus B D, Schickli J H, Blau D M, et al. Conformational changes in the spikeglycoprotein of murine coronavirus are induced at37degrees C either by solublemurine CEACAM1receptors or by pH8[J]. J Virol,2003,77(2):830-840.
[94] Wentworth D E, Holmes K V. Addition of a single glycosylation site to hAPNblocks humancoronavirus-229E receptor activity [J]. Adv Exp Med Biol,2001,494(199-204.
[95] Wentworth D E, Holmes K V. Molecular determinants of species specificity inthe coronavirus receptor aminopeptidase N (CD13): influence of N-linkedglycosylation [J]. J Virol,2001,75(20):9741-9752.
[96] Krempl C, Schultze B, Herrler G. Analysis of cellular receptors for humancoronavirus OC43[J].Adv Exp Med Biol,1995,380(371-374.
[97] Popova R, Zhang X. The spike but not the hemagglutinin/esterase protein ofbovine coronavirus is necessary and sufficient for viral infection [J]. Virology,2002,294(1):222-236.
[98] Li W, Moore M J, Vasilieva N, et al. Angiotensin-converting enzyme2is afunctional receptor for the SARS coronavirus [J]. Nature,2003,426(6965):450-454.
[99] Hofmann H, Pohlmann S. Cellular entry of the SARS coronavirus [J]. TrendsMicrobiol,2004,12(10):466-472.
[100] de Haan C A, Stadler K, Godeke G J, et al. Cleavage inhibition of the murinecoronavirus spike protein by a furin-like enzyme affects cell-cell but notvirus-cell fusion [J]. J Virol,2004,78(11):6048-6054.
[101] Gombold J L, Hingley S T, Weiss S R. Fusion-defective mutants of mousehepatitis virus A59contain a mutation in the spike protein cleavage signal [J]. JVirol,1993,67(8):4504-4512.
[102] Leparc-Goffart I, Hingley S T, Chua M M, et al. Targeted recombination withinthe spike gene of murine coronavirus mouse hepatitis virus-A59: Q159is adeterminant of hepatotropism [J]. J Virol,1998,72(12):9628-9636.
[103] Hingley S T, Leparc-Goffart I, Weiss S R. The spike protein of murinecoronavirus mouse hepatitis virus strain A59is not cleaved in primary glial cellsand primary hepatocytes [J]. J Virol,1998,72(2):1606-1609.
[104] Greig AS, Mitchell D, Corner AH, Bannister GL, Meads EB, Julian RJ (1962) AHemagglutinating Virus Producing Encephalomyelitis in Baby Pigs. Can J CompMed Vet Sci26(3):49-56.
[105] Pensaert MB, Callebaut PE (1974) Characteristics of a coronavirus causingvomition and wasting in pigs. Arch Gesamte Virusforsch44(1):35-50.
[106] Sasseville AM, Gelinas AM, Sawyer N, Boutin M, Dea S (2001) Biological andmolecular characteristics of an PHEV isolate associated with recent acuteoutbreaks of encephalomyelitis in Quebec pig farms. Adv Exp Med Biol494:57-62.
[107] Chang GN, Chang, T. C., Lin, S. C., Tsai, S. S., Chern, R. S.(1993) Isolationand identification of hemagglutinating encephalomyelitis virus from pigs inTaiwan. J Chin Soc Vet Sci19:147-158
[108] Chen KY, He, W. Q., Lu, H. J., Song, D. G., Gao, W., Lan, Y. G., Zhao, K.&Gao, F.(2011) Development of an immunochromatographic strip for serologicaldiagnosis of Porcine hemagglutinating encephalomyelitis virus. J Vet DiagnInvest23:In press
[109]贺文琦,陆慧君,耿百成,宋德光,姜宁,Hirano N,高丰,等.猪血凝性脑脊髓炎病毒抗体的调查.中国兽医科技,2005,35(9):739-741.
[110]耿百成,高丰,贺文绮,等.血凝性脑脊髓炎病毒血凝抑制试验方法的研究[J］.动物医学进展,2005,26(6):98-100.
[111]姚龙涛主编.猪病毒病[M].上海科学出版社,2000.
[112]徐耀先,周晓峰,刘立德.动物病毒分子生物学[M].武汉:湖北科学技术出版社,2000.
[113]莱曼AD.猪病学第6版[M].北京:农业大学出版社,1990.
[114]高丰,丁壮,安铁洙,等.不同途径感染猪血球凝集性脑脊髓炎病毒的比较病理学[J].中国兽医学报,2003,4(23):366-368.
[115]常灵竹,贺文琦,陆慧君.等.猪血凝性脑脊髓炎病毒RT-PCR方法的建立及初步应用[J].中国农学通报.2007.23(9):15-18.
[116]藏德跃,王栋，李明谦,等.猪血凝性脑脊髓炎病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立[J].中国兽医科学,2011,41(1):60-64.
[117]丁宁.猪血凝性脑脊髓炎病毒LAMP检测方法的建立及其临床初步应用
[D].2012.
[118]赵传博,王丽,董波,等.猪血凝性脑脊髓炎病毒抗原捕获ＥＬＩＳＡ诊断方法的建立[J].中国兽医学报,2013,33(4):526-531.
[119] Chakraborti, S., Prabakaran, P., Xiao, X., Dimitrov, D.S.,2005. The SARScoronavirus S glycoprotein receptor binding domain:fine mapping and functionalcharacterization.
[120] He, Y., Zhou, Y., Liu, S., Kou, Z., Li, W., Farzan, M., Jiang, S.,2004a.Receptor-binding domain of SARS-CoV spike protein induces highlypotent neutralizing antibodies implication for developing subunit vaccine.Biochem. Biophys. Res. Commun.324,773–781.
[121]孙宇,贾凌云,任军.蛋白质相互作用的研究方法[J].分析化学评述与进展,2007,35(5):760-766.
[122]陈谋通,刘建军.蛋白质相互作用的研究方法.生物技术通报,2009,1:50-54.
[123]夏其昌,曾嵘.蛋白质化学与蛋白质组学.北京:科学出版社,2004,456-462
[124] Saito S, Liu F, Kamijo K, et al． Deregulation and mislocalization of thecytokinesis regulator ECT2activate the Rho signaling pathways leading tomalignant transformation [J]． J Biol Chem,2004,279(8):7169－7179．
[125] V IKIS H G,GUAN K L.Glutathione-S-transferase-fusion based assays forstudying protein-protein interactions.Methods Mol Biol,2004,261:175-186
[126] Chiang S, Hwang J, Legendre M, et al． TCGAP, a multidomain Rho GTPase－activating protein involved in insulin－stimulated glucose transport[J]．EMBO J,2003,22(11):2679－2691．
[127] Bi Y,Williams A． A role for Rho and Rac in secretagogue induced amylaserelease by pancreatic acini [J]． Am J Physiol,2005,289(1):C22－32．
[128] Ridley J, Paterson H, Johnston C, et al． The small GTP－binding protein racregulates growth factor－induced membrane ruffling[J]． Cell,1992,70(3):401－410．
[129]侯佩莉. GST pul-l down验证流感病毒PB1-F2蛋白与热休克蛋白40的相互作用[J].生物技术通报,2011,1:202-205.
[130]李青岭.一个约30kD膜蛋白作为汉坦病毒候选受体的筛选[J].生物技术通报,2008,4:125-129.
[131]于莉娜,钟雪云,陈运贤,等.免疫共沉淀法用于肿瘤异质性研究的可行性分析.免疫学杂志,2004,20(1):58-64.
[132] Li WH, Moore MJ, Vasillieva N, et al．Angiotension converting enzyme2is afunctional receptor for SARS coronavirus [J]. Nature,2003,426(6965):450-454.
[133]刘美德,赵彤言等. C6/36细胞上登革2型病毒受体的免疫共沉淀[J].中国病毒学.2004,19(3):217-219.
[134] Tabassum R, Chavali S, Dwivedi O P, et al． Genetic variants of FOXA2: riskof type2diabetes and effect on metabolic traits in North Indians[J]． J HumGenet,2008,53(11-12):957-965．
[135] Ivorra C, Kubicek M, González J M, et al． A mechanism of AP-1suppressionthrough interaction of c－Fos with lamin A/C [J]． Genes Dev,2006,20(3):307-320．
[136] Hanane Boukarabila, Andrew J. S, Eric Batsche, et a.l The PRC1Polycombgroup complex interactswith PLZF/RARA to mediate leukem ic transformation[J]. Genes Dev,2009,23(10):1195-1206.
[137]张艳玲,张宏,侯平,等.新基因AngReml04与糖皮质激素受体特异延伸因子的相互作用[J].中华肾脏病杂志,2004,20(4):246-249.
[138]吴小成,何水珍,郑子峥,等. HepG2细胞中与戊型肝炎病毒衣壳蛋白相互作用蛋白的初步研究[J].病毒学报,2006,(22):329-333.
[139] Zeki T,Katherine L,Borden B.The yeast two-hybrid system and itspharmaceutical significance. Pharmaceutical Research,2000,Vol(17):9
[140]马洪波,杜坚.酵母双杂交系统的研究进展与应用.中国国境卫生检疫杂志,2004,27(2):119-123.
[141]王玉娥,杨汉春.酵母双杂交技术在病毒学研究中的应用.中国兽医学报,2004,24(3):307–310.
[142]吴志聪,王一飞.酵母双杂交系统.生物技术,2003,13(5):43-44.
[143]吴娟,钱凯,杨泽峰.酵母双杂交系统的研究进展.安庆师范学院学报(自然科学版),2005,1l (2):59-63.
[144] Yang M,Wu Z,Fields S.Protein-peptide interactions analyzed with the yeasttwo-hybrid system.Nucleic Acids Res,1995,23:l152-1156.
[145] CHO S,PARK S G,LEE D H,et al.Protein-protein interaction net-works:frominteractions to networks.J Biochem Mol Biol,2004,37(1):45-52.
[146] STEPHEN D J,BANTING G.The use of yeast two-hybrid screens in studies ofprotein:Protein interaction involved in trafficking.Traffic,2009(10):763.
[147] HIRSTM,HO C,SABOURIN L,et al.A two-hybrid system for trans-activatorbait proteins. Proc Natl Acad Sci USA,2001,98(15):8726-8731.
[148] Fields S,Song O.A novel genetic system to detect protein-proteininteractions.Nature,1989,340:245-246
[149] Rual J F,Venkatesan K,Hao T,et al.Towards a proteome-scale map of the humanprotein-protein interaction network.Nature,2005,437(7062):1173-1178
[150]朱金鑫,李小方.酵母双杂交技术及其在植物研究中的应用.植物生理学通讯,2004,40(2):235-240.
[151] ARONHEIM A,ZAND I E,HENNEMANN H,et al.Isolation of an AP-1repressor by a novel method for detecting protein-protein interactions.Mol CellBiol,1997,17(6):3094-3102
[152]夏其昌,曾嵘.蛋白质化学与蛋白质组学.北京:科学出版社,2004,456-462.
[153] Drewes G,Bouwmeester T. Curr Opin Cell Biol,2003,15(2):199-205.
[154] Bauer A,Kuster B. Eur J Biochem,2003,270(4):570-578.
[155]. Matchmaker Library Construction＆Screening Kit User Manual.2003:33-34.
[156]李凌云,刘鑫,张鹏,等.麻疹病毒绒猴细胞受体基因的克隆与功能鉴定[J].科学通报.2002,16(47):1217-1225.
[157] M Vidal, R K Brachmann, A Fattaey, et a1. Reverse two-hybrid and one-hybridsystems to detect dissociation of protein-protein and DNA-protein interactions[J]. Proc Natl Acid Sci,1996,93:10315-10320.
[158] Aronheim A, Zandi E, Hennemann H, et al. Isolation of an AP-1represso by anovel method for detecting protein-protein interactions[J]. Mol Cell Biol,1997,17:3094-3102.
[159]张素华,钱程,吴海玮.噬菌体cDNA展示文库技术及其在寄生虫学中的应用[J].国际医学寄生虫病杂志,2007,34(2):103-107.
[160]帅武平,高建青,陈海靓,等.噬菌体表面展示技术及其在靶向载体构建中的应用[J].中国医药杂志,2007,42(4):246-249.
[161] Paul P Sche, Kathleen M Mckenzie, et a1. Display cloning: fuctionalidentification of natural product receptors using cDNA-phage display[J].Chemistry＆Biology,1999,6:707-716.
[162]董倩,施双双,王业东,等. cDNA文库噬菌体展示法的建立及乙型肝炎病毒前S1蛋白结合蛋白筛检[J].解放军医学杂志.2002,4(27):321-322.
[163]白雪帆，张英,汪毅．应用PEPSCAN技术研究单克隆抗体L13F3识别的抗原表位．第四军医大学学报，2000，21（7）：850-852．
[164] Hideo G., Nobuyuki M., Hiroshi I. Mapping of epitopes and structural analysisof antigenic sites in the nucleoprotein of rabies virus. J Gen Virol.2000,81:119-127.
[165] Sorkin M.M., Huang F., Carter R. Interaction of EGF receptor and Grb2inliving cells visualizedby fluorescence resonance energy transfer (FRET)microscopy. Curr Biol.2000,10(21):1395-1398.
[166]黄翠芬,叶棋浓.蛋白质间相互作用技术的研究近况[J].中国生物化学与分子生物学报,1998,14(1):125-128.
[167] Meyer G., Novel N.M. Optical approach to atomic force microscopy. Appl PhysLett.1988,53(12):1045-1047.
[168] Legrand D., Vigié K., Said E.A., Elass E., Masson M., Slomianny M.C.,Carpentier M., Briand J.P.,Mazurier J., Hovanessian A.G. Surface nucleolinparticipates in both the binding and endocytosis of lactoferrin in target cells. EurJ Biochem.2004,271(2):303-317.
[179] Liu S, Xiao G, Chen Y, He Y, Niu J, Escalante CR, Xiong H, Farmar J, DebnathAK, Tien P, Jiang S. Interaction between heptad repeat1and2regions in spikeprotein of SARS-associated coronavirus: implications for virus fusogenicmechanism and identification of fusion inhibitors. Lancet2004;363(9413):938-47.
[170] Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, SomasundaranM, Sullivan JL, Luzuriaga K, Greenough TC, Choe H, Farzan M.Angiotensin-converting enzyme2is a functional receptor for the SARScoronavirus. Nature2003;426(6965):450-4.
[171] Wei Gao, Wenqi He, Kui Zhao, Huijun Lu, Wenzhi Ren, Chongtao Du,Keyan Chen, Yungang Lan,Deguang Song, Feng Gao. Identification of NCAMthat interacts with the PHE-CoV spike protein.Virlogy Journal.2010,7:254.
[172] Wong SK, Li W, Moore MJ, Choe H, Farzan M. A193-amino acid fragment ofthe SARS coronavirus S protein efficiently binds angiotensin-converting enzyme2. J. Biol. Chem,2004..279:3197-3201.
[173] Kubota S., Sasaki O., Amimoto K., Okada N., Kitazima T., Yasuhara H.Detection of porcine epidemic diarrhea virus using polymerase chain reactionand comparison of the nucleocapsid protein genes among strains of the virus. JVet Med Sci.1999,61(7):827-830.
[174] Du L, Zhao G, Kou Z, Ma C, Sun S, Poon VK, Lu L, Wang L, DebnathAK, Zheng BJ, Zhou Y, Jiang S. Identification of a receptor-binding domain inthe s protein of the novel human coronavirus Middle East respiratorysyndrome coronavirus as an essential target for vaccine development. JVirol.2013,87(17):9939-42.
[175] Leparc-Goffart I, Hingley S T, Chua M M, et al. Altered pathogenesis of amutant of the murine coronavirus MHV-A59is associated with a Q159L aminoacid substitution in the spike protein[J]. Virology,1997,239(1):1-10.
[176] Thackray L B, Holmes K V. Amino acid substitutions and an insertion in thespike glycoprotein extend the host range of the murine coronavirus MHV-A59[J]. Virology,2004,324(2):510-524.
[177] Li W, Zhang C, Sui J, et al. Receptor and viral determinants ofSARS-coronavirus adaptation to human ACE2[J]. EMBO J,2005,24(8):1634-1643.
[178] Lai S C, Chong P C, Yeh C T, et al. Characterization of neutralizing monoclonalantibodies recognizing a15-residues epitope on the spike protein HR2region ofsevere acute respiratory syndrome coronavirus (SARS-CoV)[J]. J Biomed Sci,2005,12(5):711-727.
[179] Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular EvolutionaryGenetics Analysis (MEGA) software version4.0. Molecular biology andevolution24(8):1596-1599.
[180] Follis K.E., York J., Nunberg J.H. Furin cleavage of the SARS coronavirusspike glycoprotein enhances cell–cell fusion but does not affect virion entry.Virology,2006,350(2):358-69.
[181]张嘉杰.SARS冠状病毒S蛋白抗原表位多肽的设计、合成及其免疫反应[D].第一军医大学
[182] KunkelF,HerrlerG Structural and functional analysis of the surfaee Protein ofhuman coronavirus OC43.Virology,1993,195:195-202
[183]姜愚，文艳君，刘继彦，等.小鼠B细胞趋化因子原核农达与纯化[J].生物医学工程学杂志，2004,21(2):251-255.
[184] Hefti MH, Van Vugt-Van der Toorn CJ, Dixon R, et al. A novel purificationmethod for histidine-tagged proteins containing a thrombin cleavage site[J]. AnalBiochem,2001,295(2):180-185.
[185] Babcock G.J., Esshaki D.J., Thomas W.D., Ambrosino D.M. Amino acids270to510of the severeacute respiratory syndrome coronavirus spike protein arerequired for interaction with receptor. J Virol.2004,78(9):4552-4560.
[186] Bonavia A., Zelus B.D., Wentworth D.E., Talbot P.J., Holmes K.V.Identification of a Receptor-Binding Domain of the Spike Glycoprotein ofHuman Coronavirus HCoV-229E. J Virol.2003,77(4):2530-2538.
