AIV（H9N2）NS1蛋白单抗的研制及单抗介导ELISA对AIV（H9N2）HA抗原表位变异研究
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摘要
禽流感病毒(Avian Influenza Virus，AIV)的非结构蛋白(NS1)可作为一种区分野毒感染和灭活疫苗免疫鸡群的鉴别诊断标记，但不能区分AIV亚型，具有A型流感特异性。本文利用本河南农业大学禽病研究所实验室已构建的含有H9N2亚型AIV NS1蛋白基因的重组表达载体阳性菌，在37℃条件下，用终浓度1mmol／L的IPTG诱导表达5h，经15％的SDS-PAGE分析，表明NS1基因在大肠杆菌中得到高效表达。用HisTrap HP Kit进行复性和纯化，获得了纯度较高、生物活性较好的NS1蛋白。以此NS1蛋白作为抗原免疫Balb／c小鼠，应用杂交瘤技术，通过ELISA筛选，建立了2株抗H9N2亚型AIV NS1单克隆抗体(Monoclonal Antibody，McAb，简称单抗)杂交瘤细胞株689和6C2，其分泌单抗抗能力稳定。将2株杂交瘤细胞株分别注射小鼠收获的腹水单抗效价分别为100×212和100×210。制备的单抗有良好的热稳定性和较高的特异性，只与AIVNS1蛋白反应，而与H5N2亚型AIV、H9N2亚型AIV、NDV、IBV、IBDV、ILTV、EDS76V均不发生反应。NS1蛋白单抗的成功研制为利用该单抗开发一种快速、特异、敏感的鉴别野毒感染禽群和疫苗免疫禽群的方法及进一步研制诊断试纸条或组装成试剂盒奠定了基础，也为深入研究NS1蛋白的结构和功能奠定了良好的基础，对AI的早期诊断、适时监控和净化具有重要现实意义。
目前H9亚型AI仍是我国的流行主型，由于AIV抗原性和致病力的易变异性及不同血清型毒株间缺乏交叉保护性，加大了AI防制的难度。本试验将本实验室已建立的4株抗H9N2亚型AIV血凝素(HA)单克隆抗体与保存的1998-2005年间的25株H9N2亚型AIV毒株进行交叉ELISA试验，发现不同H9N2亚型AIV毒株血凝素(HA)抗原表位及毒力有明显差异，可将25株H9N2型AIV毒株分为4类，进一步证实了不同H9N2型AIV毒株HA抗原表位和毒力的变异性和多态性。其中2株毒株(A3和A18)比较特殊，与4株H9N2亚型AIV HA单抗均不发生反应，缺少4株单抗所识别的HA抗原表位，但其毒力很强。对25株H9N2亚型AIV毒株进行深入研究将对H9N2型AIV的防制在理论和实践上均具有一定的指导意义。
Nonstructural genel (NS1) of AIV (H9N2) was cloned into the pET-28(a) vector to construct a recombinant expression plamid.After the recombinant expression plasmid was transform into E.coli ,the recombinant protein was induced by 1mmol/L IPTG at 37°C for 5 hours and analyzed by SDS-PAGE. The result showed that NS1 protein was highly expressed in a way of inclusion body. Its molecular weight was 28KD as expected. The expression NS1 protein was denatured and refolded and purified by HisTrap HP Kit. The result indicated that the high pure and active NS1 protein was obtained.
Eight-week Balb/c mice were immunized by NS1 protein according to Kazuhiro's produre.Two hybridoma were generated by fusion of SP2/0 myelomas and spleen cells from immunized mice and screened by ELISA and named 6B9、 6C2 respectively. The monoclonal antibodies(McAbs) were produced and proved to be IgG1、IgG2b by AGP.Mouse ascetic fluid was purified (protein concentration: 19mg/ml、14.9mg/ml) and proved to react with NS1 protein of AIV (H9N2)by HI and ELISA. The ELISA or rapid diagnosis strip which was based on the McAbs will provide a kind of sensitive, specific differentiating diagnostic approach and an effective method for early diagnosis, real-time detection and eradication of AIV.
In order to study the variation of antigenicity in HA of AIV (H9N2), twenty-five AIV (H9N2) strains of China during 1998-2005 were detected by ELISA based on four McAbs specific to hemagglutinin (HA) of AIV (H9N2). The results of ELISA demonstrated that antigenicity and virulence had variated in the virus strains during 1998-2005, the antigenic site and virulence of A3 and A18 strain among them had become stronger. The results of our study would provide scientific reference for prevention against AIV and selection of vaccine strains.
目前H9亚型AI仍是我国的流行主型，由于AIV抗原性和致病力的易变异性及不同血清型毒株间缺乏交叉保护性，加大了AI防制的难度。本试验将本实验室已建立的4株抗H9N2亚型AIV血凝素(HA)单克隆抗体与保存的1998-2005年间的25株H9N2亚型AIV毒株进行交叉ELISA试验，发现不同H9N2亚型AIV毒株血凝素(HA)抗原表位及毒力有明显差异，可将25株H9N2型AIV毒株分为4类，进一步证实了不同H9N2型AIV毒株HA抗原表位和毒力的变异性和多态性。其中2株毒株(A3和A18)比较特殊，与4株H9N2亚型AIV HA单抗均不发生反应，缺少4株单抗所识别的HA抗原表位，但其毒力很强。对25株H9N2亚型AIV毒株进行深入研究将对H9N2型AIV的防制在理论和实践上均具有一定的指导意义。
Nonstructural genel (NS1) of AIV (H9N2) was cloned into the pET-28(a) vector to construct a recombinant expression plamid.After the recombinant expression plasmid was transform into E.coli ,the recombinant protein was induced by 1mmol/L IPTG at 37°C for 5 hours and analyzed by SDS-PAGE. The result showed that NS1 protein was highly expressed in a way of inclusion body. Its molecular weight was 28KD as expected. The expression NS1 protein was denatured and refolded and purified by HisTrap HP Kit. The result indicated that the high pure and active NS1 protein was obtained.
Eight-week Balb/c mice were immunized by NS1 protein according to Kazuhiro's produre.Two hybridoma were generated by fusion of SP2/0 myelomas and spleen cells from immunized mice and screened by ELISA and named 6B9、 6C2 respectively. The monoclonal antibodies(McAbs) were produced and proved to be IgG1、IgG2b by AGP.Mouse ascetic fluid was purified (protein concentration: 19mg/ml、14.9mg/ml) and proved to react with NS1 protein of AIV (H9N2)by HI and ELISA. The ELISA or rapid diagnosis strip which was based on the McAbs will provide a kind of sensitive, specific differentiating diagnostic approach and an effective method for early diagnosis, real-time detection and eradication of AIV.
In order to study the variation of antigenicity in HA of AIV (H9N2), twenty-five AIV (H9N2) strains of China during 1998-2005 were detected by ELISA based on four McAbs specific to hemagglutinin (HA) of AIV (H9N2). The results of ELISA demonstrated that antigenicity and virulence had variated in the virus strains during 1998-2005, the antigenic site and virulence of A3 and A18 strain among them had become stronger. The results of our study would provide scientific reference for prevention against AIV and selection of vaccine strains.
引文
[1] 甘孟侯．禽流感(第二版)[M]．北京：中国农业大学出版社，2002
[2] B W卡尔尼克．禽病学(第十版)[M]．北京：中国农业大学出版社，1999
[3] 甘孟侯．中国禽病学[M]．北京：中国农业出版社，1999．7
[4] Oxford J S. Influenza pandemics of the 20th century with special reference to 1918: Virology, pathology and epidemiology [J]. Rev Med Virol, 2000, 10(2): 119-133
[5] 甘孟侯．全球禽流感的流行形势[J]．中国预防兽医学报，2004，26(6)：468-471
[6] Alexander D J. A reviews of avian influenza in diferent bird species [J]. Vet Microbiol, 2000, 74(1-2): 3-13
[7] 殷震，刘景华．动物病毒学(第二版)[M]．科学出版社，1997
[8] Lu X H, Cho D. Pathogenesis of and immunity to a new influenza A (H5N1) virus isolated from duck meat [J]. Avian Dis, 2003, 47(3): 1135-1140
[9] Tumpey T M. Evaluation of a hipathogenicityl15Nl avian influenza A virus isolated fromduck meat [J]. Avian Dis, 2003, 47(3): 951-953
[10] Baigent, S J, J W. McCauley. Glycosylation of hemagglution and stalk-length of neuraminidase combine to regulate the growth of avian influenza virus in tissue culture [J]. Virus Research, 2001, 79: 177-185
[11] 黄庚明，辛朝安．PCR制备地高辛标记的探针检测禽流感病毒核酸[J]．中国兽医杂志，2001，37(12)：3-7
[12] 郑腾，陈枝华．禽流感的公共卫生意义[J]．生物学通报，2002，37(4)：13-15
[13] OIE. Disease Information [J].2002, 15(29): 1-8
[14] Donateli I, Campiteli L. Characterization of H_5N_2 influenza virus from Italian poultry [J]. J Gene Virol, 2001, 82(3): 623-630
[15] 范伟兴，何海权，腾翔雁，等．近期亚洲高致病性AI流行态势简析[J]．2004，21(5)：46-48
[16] Home P J, Easterday B C. Avian influenza virus infections: Response of pheasants, ducks and geese to influenza A-turkey-Wisconsin-1966 virus [J]. Avian Dis, 1970, 14(2): 285-290
[17] 郭元吉．流感病毒生态学研究近况[J]．流行病学杂志，1980，1(1)：61-65
[18] 陈伯伦，张泽纪，陈伟斌，等．鸡A型AIV的分离与血清学初步鉴定[J]．中国兽医杂志，1994，20(10)：3-5
[19] 陈福勇，夏春．AIA／鸡／北京／1／96(H9N2)株核蛋白基因克隆和序列分析[J]．中国预防兽医学报，1999，21(2)：130-133
[20] 王永坤．鸭流感防治研究进展[J]．辽宁畜牧兽医，2003，6：32-36
[21] 席瑞珍．鸭源H_5N_1亚型AIV与鸡源H_5N_1亚型AIV的生物学特性和分子生物学特性的比较[D]．河南农业大学硕士论文，2004．6
[22] Hinshaw V. S, Bean W J, Geraci J R, et al. Characterization of two influenza A viruses from a pilot whale[J]. J. Virol, 1986, 58: 655-656
[23] Kingeborn B L, Englund R, Rott N, et al. An avian influenza A virus killing a mammalian species—the mink[J]. Arch. Virol, 1985, 86: 347-351
[24] 李雁冰，田国彬，李艳华，等．不同宿主的H_5亚型AIV对小鼠的致病性试验[A]．中国畜牧兽医学会家畜传染病学分会第十次学术研讨会禽流感研究论文专集，2003．10
[25] 焦培荣，李泽君，侯勇跃，等．四株H_5亚型AI对小鼠致病性的研究[A]．中国畜牧兽医学会家畜传染病学分会第十次学术研讨会禽流感研究论文专集，2003．10
[26] 乔传玲，于康震．AIV结构蛋白及其免疫原性[J]．预防兽医学进展，2000，2(4)：4-7
[27] 刘金华，史为民，吴清民，等．我国鸡源与人源H_9N_2亚型AI聚合酶PB_1基因关系分析[J]．中国人兽共患病杂志，2004，20(7)：589-591
[28] Yao Y, Mingay L J. Sequences in influenza virus PB_2 protein that determine productive infection for an avian influenza virus in mouse and human cell line[J]. Arch Virol, 2001, 130(12): 209-217
[29] Massin P, Vander W S. Residue 627 of PB_2 is a determinant of cold sensitivity in RNA raplicatinn of avian influenza virus in mouse and human cell line[J]. J Virol, 2001, 75(11): 5398-5404
[30] 黄庚明，辛朝安．AIV血凝素(HA)基因的研究进展[J]．广东畜牧兽医科技，2001，26(2)：3-5
[31] 李玉清，王宁．流感的分子流行病学研究进展[J]．疾病控制杂志，2001，5(1)：60-62
[32] Tonegawa K, Nobusawa E, Nakaja K, etal. Analysis of epitope recognition of antibodies induced by DNA inunization against hemagglutinin protein of influenza A virus[J]. Vaccine, 2003, 21(23): 3118-3125
[33] 吴清民．AIV核蛋白基因表达的研究[D]．中国农业大学博士论文，2002
[34] 金元昌，李景鹏，张龙，等．AIV分子生物学研究进展[J]．动物医学进展，2003，24(1)：12-15
[35] 卢建红，韦栋平，刘玉良，等．H_9N_2亚型AI核蛋白基因克隆及其序列特征[J]．中国兽医科技，2003，4：46-49
[36] 何后军，戴益民，付光华，等．AIV分子生物学进展[J]．江西农业大学学报，2004，26(2)：290-293
[37] 刘菲，郭宇飞，冉智光，等．AIV的分子生物学基础和疫苗控制研究[J]．中国家禽，2004，8(1)：195-198
[38] 刘洪雨，刘玉芬，于尔松，等．AIV的研究[J]．畜牧兽医科技信息，2005，1：4
[39] 石火英，刘秀梵．利用反向遗传技术研究H_9N_2亚型AIV传播途径的分子机制[J]．微生物学报，2006，
[40] 郭元吉，温乐英，王敏，等．我国猪群中H_9N_2亚型毒株卧和NA基因特性的研究[J]．中华试验和临床病毒学杂志，2004，18(1)：7-11
[41] 乔传玲，张建林，贾永清，等．禽流感病毒血凝素与核蛋白在重组禽痘病毒中的共表达[J]．动物医学进展，2003，24(1)：81—83
[42] 褚玲娜，詹勇，许梓荣，等．AIV的致病性和免疫性研究进展[J]．中国兽药杂志，2005，39(1)：28-32
[43] Liu W, LiH, Chen Y H. N-terminus of M2 protein could induce antibodies with inhibiitory activity against influenza virus replication[J]. FEMS lmmunol Med Microbio, 2003, 35 (2): 141—146
[44] 王传彬，孙明，赵铁柱，等．H9N2AIV分离株NS基因同源性分析[J]．预防兽医学报，2004，26(3)：177-180．
[45] 孙明，赵铁柱，王传彬，等．AIVH5N1亚型NS1基因在大肠杆菌中的表达[J]．中国试验动物学报，2004，12(4)：208-211
[46] Lamb R A, Krug R M. Orthomyxoviridae: the viruses and their replication [J]Virology, 2001, 1487-1532
[47] 郭元吉，程小雯．流行性感冒病毒及其试验技术[M]．重庆：中国三峡出版社，1997，16-20．
[48] Cassetti M C, Noah D L, Montelione G t, et al. Efficient translation of mRNAs in influenza A virus-infected cells is independent of the viral 5' untranslated region [J]. Virology, 2001, 289(2): 180-185
[49] Li Y, Yamakita Y, Krug R M, et al. Regulation of a nuclear export signal by an adjacent inhibitory sequence: The effector domain of the influenza virus NS1 protein [J]. Proc. Natl. Acad. Sci. USh, 1998, 95: 4864-4869
[50] Yuan W, AraminiJ M, Montelione G T, et al. Structural basis for ubiquitin-like 1SG15 protein binding to the NS1 protein of influenza B virus:a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS 1 protein of influenza A virus[J]. Virology, 2002, 304(2): 291-301.
[51] Chien C Y, Xu Y, Xiao R, et al. Biophysical characterization of the complex between double-stranded RNA and the N-terminal domain of the NS1 protein from influenza A virus: evidence for a novel RNh-binding mode[J]. Biochemistry, 2004,43(7): 1950-1962
[52] 李先茜，杜培革，傅桂莲，等．应用定点突变技术进行流感病毒NSI蛋白功能的研究[J]北华大学学报，2004，5(4)：316-320
[53] Skehel JJ. Polypeptidel synthesis in influenza virus-infected cells[J]. Virology, 1972, 49(1):23-36
[54] Zhirnov O P, Konakova T E, wolff T, et al. NS, protein of influenza A virus down-regulates apoptosis[J].J Virol, 2002, 76(4):1617-1625
[55] Geiss G K, Salvatore M, Tumpey T M, et al. Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: The role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza[J]. Proc Natl Acad Sci USA, 2002, 99(16): 10736-10741.
[56] Seo S H, Webster R G. Tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells [J]. J Virol, 2002, 76(3): 1071-1076.
[57] Seo S H, Hoffmann E, Webster R G, et al. Lethal H5N1 influenza viruses escape host antiviral cytokine responses [J]. Nature Med, 2002, 8(9): 950-954.
[58] Basler C F, Reid A H, Dybing J K, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes[J]. Proc. Natl. Acad. Sci. USA, 2001 27; 98(5): 2746-51
[59] TaubenbergerJ K, Reid A H, Janczewski T A, et al. Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus[J]. Philos Trans SocLond Biol Sci, 2001,356(1416):1829-39.
[60]Qiu Y, Krug R M. The influenza virus NS1 protein is a poly (A)-binding protein that inhibits nuclear export of mRNAs containing poly(A)[J]. J. Virol, 1994, 68(4):2425-2432.
[61]Chen Z, Li Y.Krug RM, et al. Influenza A virus NS1 protein targets poly(A)-binding protein of the cellular 3' end processing machinery [J]. EMBO J, 1999, 18(8) :2273 - 2283
[62]Chen ,Krug R M. Selective nuclear export of viral mRNAs in influenza virus infected cells[J]Trends Microbiol, 2000, 8:376-383.
[63]Lu Y, Qian X Y, Krug R M, et al.The influenza virus NS1 protein:a novel inhibitor of pre-mRNA splicing[J]. Genes, 1994, 8:1817-1828
[64]Fortes P, Beloso A.Ortin J, et al. Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport[J]. EMBO J, 1994,13:704-712
[65]Nemeroff M E, Barabino S M, Li Y, et al. Influenza virus NS1 protein interacts with the cellular 30 KD subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs[J].Mol Cell,1998,1:991-1000
[66]Enami K, Sato T A, Nakada S, et al. Influenza virus NS1 protein stimulates translation of the M_1 protein[J].J Virol,1994,68:1432;1437
[67]Noah D L, Tkuky, Krug R M, et al. Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS_1 A protein via its binding to a cellular protein required forthe 3' end processing of cellular pre-mRNAs[J]. Virology, 2003, 307(2):386-395.
[68]Schultz S, Dybdahl-Sissoko N, Neumann G, et al. Influenza virus NS1 protein induces apoptosis in cultured cells[J].J Virol, 2001, 75(17):7875-7881
[69]Takizawa, Lowy R J. Influenza virus induction of apoptosis by intrinsic and extrinsic mechanisms[J]. Int Rev Immunol, 2003, 22(5-6):425-49
[70]Zhirnov O P Konakova T E, wolff T, et al. NS_1 protein of influenza A virus down regulates apoptosis[J].J Virol, 2002, 76(4):1617-1625.
[71]Lu Y, Wambach, Katze M G, et al. Binding of the influenza virus NS1 protein to double stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor[J]. Virology, 1995 , 214 (1):222-228
[72]Garcia-Sastre A, Egorov D, Matassov, et al. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems[J]. Virology, 1998, 252(2):324-330
[73]Talon J, Salvatore M, Neill R E, et al. Influenza A and B viruses expressing altered NS1 proteins:a vaccine approach[J]. Nat1 Acad Sci USA, 2000, 97(8):4309-4314
[74]Stasakova J, Ferko B, Kittel C, et al. Influenza A mutant viruses with altered NS1 protein function provoke caspase-1 activation in primary human macrophages, resulting in fast apoptosis and release of high levels of interleukins beta [J]. J Gen Virol, 2005, 86(Pt1): 185-195.
[75] Ferko B, Stasakova J, Romanova J, et al. Immunogenicity and protection efficacy of replication-deficient influenza A viruses with altered NS1 genes[J]. J Virol, 2004, 78(23): 13037-13045
[76] Wang X M, Li, H, Zheng, et al. Influenza A virus NS1 protein prevents the activation of NF-κB and induction of type I IFN[J]. J. Virol, 2000, 74(24): 11566-11573
[77] Kazufumi S, Kazumichi K. Expression of host genes in influenza virus infected cells [J]. Uirusu., 2004, 54(2): 189-96
[78] Zurcher Y, Marion R M,Ortin J, et al. Protein synthesis shut-off induced by influenza virus infection is independent of PKR aetivity[J]. J Virol, 2000, 74: 8781-8784
[79] Bergmann M, Garcia-Sastre A. Influenza virus NS1 protein counteracts PKR-mediated inhibition of replieation[J]J. Virol, 2000, 74(13): 6203-6206
[80] Stephan, Ludwig, Xiuyan, et al. Yhe Influenza A Virus NS1 Protein Inhibits Activation of Jun N-Terminal Kinase and AP-1 Transcription Faetors[J]. Journal of Virology, 2002, 76(21): 11166-11171
[81] Geiss G K, Salvatore M, Tumpey T M, et al. Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: The role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza[J]. Proc Natl Acad Sci USA, 2002, 99(16): 10736-10741
[82] Bergmann I E, Mello P A, Neitzert E, et al. Diagnosis of persistent aphthovirus infection and its differentiation from vaccination response in cattle by use of enzyme linked immunoelectrotransfer blot analysis with bioengineered nonstructural viral antigens[J].J Vet Res, 1993, 54(6): 825-831.
[83] Rodriguez A, Dopazo J, Saiz J C, et al. Immunogenicity of non-structural proteins of foot-and-mouth disease virus: differences between infected and vaccinated swine[J]. ArchVirol, 1994, 136: 123-131
[84] Diego D M, Brocchi E, Mackay D, et al. The non-structural polyprotein 3ABC of foot-and-mouth virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle[J]. ArchVirol, 1997, 142: 2021-2033.
[85] Malirat V, Neitzert E, Bergmann I E, et al. Detection of cattle exposed to foot-and-mouth disease virus by means of an indirect ELISA test using bioengineered nonstructural polyprotein 3ABC[J]. Vet Q, 1998, 20(2): 24-26.
[86] Bergmann I E, Malirat E V, Neitzert E, etal. Improvement of a serodiagnostic strategy for foot-and-mouth disease virus surveillance in cattle under systematic vaccination: a combined system of an indirect ELISA-3ABC with anenzyme-linked immunoelectrotransfer blot assay [J]. ArchVirol, 2000, 145: 473-489.
[87] 王宏伟，孙明，田克恭，等．口蹄疫病毒3ABC、3AB、3BC基因克隆、表达及其抗体消长规律的研究[J]．中国农业科学，2004，37(7)：1065-1070
[88] 朱彩珠，尤永进，葛艳，等．用3AB-ELISA鉴别猪口蹄疫病毒感染与疫苗免疫猪的研究[J]．中国兽医科技，2005，35(1)：14-16
[89] 罗宝正，薄清如，王伟毅，等．口蹄疫病毒非结构蛋白3ABC基因的克隆与原核表达．农业生物技术学报，2005，13(2)：167—170
[90] 罗宝正，薄清如，陈金项，等．昆虫杆状病毒系统表达口蹄疫病毒3ABC基因．中国病毒学，2005，20(3)：303—306
[91] Silberstein E, Kaplan G, Taboga O, etal. Foot—and—mouth disease virus-infected but not vaccinated cattle develop antibodies against recombinant 3AB nonstructuralProtein [J]. Arch Virol, 1997, 142: 795—805
[92] 吕建强，陈焕春，赵俊龙，等．猪细小病毒NS1基因的克隆及表达[J]．中国兽医学报2003，23(5)：435-437
[93] 王君伟，等．鹅细小病毒NSI基因的克隆及原核表达．中国兽医杂志，2004，40(1)：10-13
[94] 杨敬，薛小平，雷迎峰，等．丙型肝炎病毒ns区全长基因在大肠杆菌中的高效表达及纯化．生物技术通讯，2005，1：13-15
[95] 李卫华，赵连三，周陶友，等．表达丙型肝炎病毒NS3抗原的重组腺病毒构建及体外表达．中国病毒学，2005，20(2)：101—104
[96] 沈成利，王弋嘉，石英，等．SARS冠状病毒多聚酶区抗原在感染诊断中的作用探讨．解放军医学杂志，2005，30(8)：720-724
[97] 张大鹏，王进，华子春．SARS病毒蛋白保守性分析．东南大学学报(医学版)，2005，24(1)：32—35
[98] Wang SJ, Boyle RH, Dearest VL, et al. Immunassay targeting nonstructural Protein 5 to differentiate West Nile virus infection from dengue and St. Louis encephalitis virus infections and from fiavivirus vaccination[J]. CLin Mcrobiol, 2003, 41(9): 4217
[99] Gzaki H, Sugiura T, Sugita S etal. Detection of antibodies to the nonstructural protein(NSI)of influenza A virus allows distinction between vaccinated and infected houses[J]. Veterinary Mcrobiolo gy, 2001, 82(2): 111—119.
[100] Birch-Machin I, Rowan A, Pick J, et al. Expression of the nonstructural protein NS1 of equine influenza A virus: detection of anti-NS1 antibody in post infection equine sera[J]. J. Virol Methods, 1997, 65: 255-263.
[101] Ozaki H, Sugiura T, Sugita S, et al. Detection of antibodies to the nonstructural protein(NS1)of influenza A virus allows distinction between vaccinated and infected horses[J]. Vet Microbiol, 2001, 82(2): 111-119
[102] 杨涛，刘明，张云，等．H_5N_1亚型高致病性AIVNS基因的克隆及序列分析[J]．中国兽医科技，2005，35(1)：5-9
[103] 张志珍，洪文珊，李康生，等．流感病毒NS1基因的克隆及原核表达载体的构建[J]．中国病毒学，2004，19(5)：449-453
[104] Tumpey T M, Alvarez R, Swayne D E, et al. Diagnostic approach for differentiating infected from vaccinated poultry on the basis of antibodies to NS1, the nonstructural protein of influenza A virus[J]. J Clin Microbiol, 2005, 43(2): 676-83.
[105] 王泽霖，李建丽，陈少渠，等．非结构蛋白NS1-ELISA检测禽流感野毒感染抗体[J]．科技导报，2006，3
[106] Kurtz J, Manvell R J, Banks J, et al. Avian influenza virus isolated from a woman with conjunctivitis[J]. Lancet, 1996, 348: 901-902.
[107] 王海燕，刘文博，刘秀梵，等．H9N2亚型A型流感[J]．中国禽业导刊，2004，21(9)：30-31
[108] 刘红旗，黄勇，程坚，等．在疫苗免疫选择压力下H9N2亚型禽流行性感冒病毒HA基因的遗传变异[J]．病毒学报，2002，18(2)：149
[109] 郭元吉，谢健屏，吴昆昱，等．流感病毒广州／333／99(H9N2)毒株基因组特性的研究[J]．中华试验和临床病毒学杂志，2002，16(2)：142
[110] Katz J M, Lu X. Pathogenesis of and immunity to avian influenza A H_5 viruses[J]. Biomed Pharmacother, 2000, 54(4): 178-187
[111] Swayne D E, Suarez D L. Highly pathogenic avian influenza virus[J]. Rev Sci Tech, 2000, 19(2): 463-82
[112] Rott R, Bosch F X, Garten, et al. The pathogenic determinant of influenza virus[J]. Veterinary Microbiology, 1992, 33: 303-310.
[113] Ogawa. T Genes involved in the virulence of avian influenza virus [J]. Virology, 1981, 113: 304-313
[114] Horimoto T, Kawaoka Y. Reverse genetics provides direct evidence for a correlation of hem agglutinin cleavability and virulence of an avian influenza A virus[J]. J. Virol, 1994, 68(5): 3120-3128
[115] Subbarao K, Chen H, Swayne D, et al. Evaluation of a genetically modified reassortant H_5N_1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics[J]. Virology, 2003, 305: 192-200
[116] Michael L, Perdue. How can a virus suddenly become very pathogenic?[J]. World poultry elsevier special, 2000, 6: 9-10
[117] Senne D A, Panigraphy R G, Webster, et al. Survey of the hemagglutinin (HA)cleavagesite as a marker of pathogenicity potential[J]. Avian Disease, 1996, 40(1): 425-437
[118] Besphanda K L, Fried V. A, Webste R. G, et al. Glycosylation affects cleavage of avian influenza virus hemagglutinin and regulates virulence[J]. J Virol, 2000, 74(3): 1443-1450
[119] Matrosovich M, Zhou N, Kawaoka Y, et al. The surface glycoproteins of H_5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties[J]. J Virol, 2003, 73(2): 1146-55.
[120] Goto H, Kawaoka Y. A novel mechanism for the acquisition of virulence by a human influenza A virus[J]Proc. Natl. Acad. Sci. USA, 1998, 95: 10224-10228
[121] Castrucci M R, Kawaoka Y. Biologic importance of neuraminidase stalk length in influenza A virus[J]. J. Virol, 1993, 67(2): 759-764
[122] Hatta M, Gao P, Halfmann P, et al. Molecular basis for high virulence of Hong Kong H_5N_1 influenza A virus[J]. Science, 2001, 293: 1840-1842
[123] Subbarao E K, London W, Mupphy B R, et al. A single amino acid in the PB_2 gene of influenza A virus is a detexm inant of host range[J]. J. Virol, 1993, 67(4): 1761-1764
[124] Chen W, Calvol P A, Malide D, et al. A novel influenza A virus mitochondrial protein that induces cell death[J]Nature Medicine, 2001,7(12): 1306-1312.
[125] Wang X, Castro A E, Castro M D, et al. Production and evaluation criteria of monoclonal antibodies to the hemagglutinin of the H7N2 subtype of avian influenza virus[J]. J Vet Diagn Invest, 2000, 12(6): 503
[126] Olsencw. Virologic and serologic surveillance for human, swine and avian influenza virus infections among pigs in the north-central United States[J]. Arch Virol, 2000, 145(7): 399-419
[127] Anirnov Iua. A common antigenic epitope in influenza A virus(H1,H2, H5, H6) Hemagglutinin [J].Vopr Viruol, 1999, 44(3): 11-15
[128] Fukushi H. Host species-related antigenic groups of avian influenza viruses possessing H4 hemagglutinin revealed by monoclonal antibodies[J]. Arch Virol, 1982, 72(93): 17-21
[129] 邵红霞，秦爱建，钱琨等．抗禽流感病毒H5亚型血凝素特异性单克隆抗体的研制[J]．中国动物检疫，2002．19(8)
[130] 秦爱建，邵红霞，钱琨，等．抗禽流感病毒和H5和H9亚型血凝素特异性单克隆抗体的研制及应用[J]．中国预防兽医学报，2003，25(3)：161
[131] 王泽霖，黄娟，马仲彬，等．斑点免疫金渗滤法快速检测禽流感病毒[A]．中国畜牧兽医学会禽病学分会第十二次学术讨论会论文集，2004，3：87-90
[132] 赵维波，陈美才，刘忠华等．抗禽流感病毒H5N1亚型单克隆抗体制备[J]．中国试验动物学报，2004，12(4)
[133] 曾艳兵，焦新安，潘志明，等．抗H9亚型禽流感病毒血凝素单克隆抗体的制备及初步鉴定[J]．细胞与分子免疫学杂志，2004．20 C6)：702～704
[134] 王海燕，刘文博，刘秀梵，等．H9亚型禽流感病毒分型单克隆杭体的研制及初步应用．中国家禽，2004，26(10)：21-27
[135] 王传彬，田新生，曹振，等．H5亚型禽流感病毒单抗-生物素捕获ELIsA的建立．中国试验动物学报，2004，12(4)：204-207
[136] 孔晶，吴艳涛，刘文博，等．禽流感病毒单克隆抗体的研制及初步应用．中国动物检疫，2004，21(10)：20-22
[137] 龙进学，郝贵杰，唐应华，等．H5N1亚型禽流感病毒血凝素单克隆抗体的制备及其应用．中国兽医科技，2005，35(6)：450—454
[138] 马仲彬．抗H9N2亚型禽流感病毒单克隆抗体杂交瘤细胞株的建立及快速检测试纸条的研制．河南农业大学硕士论文，2005，6
[139] 马秀丽，宋敏训，黄兵，等．H5亚型禽流感单克隆抗体的研制及应用．家禽科学，2005， 6：11-13
[140] 曹振．禽流感病毒H5亚型血凝素单克隆抗体的制备及捕获ELISA方法的建立[D]．中国农业大学硕士学位论文，2005，6
[141] 王凯，金宁一，鲁会军等．禽流感病毒H7亚型血凝素单克隆抗体的制备[J]．中国预防兽医学报，2005年，27(6)：456～457
[142] Zakstel'skaia LIa, Isachenko VA, Oskerko TA. Changes in the antigenic composition of virus A (H1N1) hemagglutinin in 1977-1982 detected by using monoclonal antibodies. Yopr Virusol. 1984 Jan-Feb; 29(1): 62-6.
[143] Yamada A, Brown LE, Webster RG. Characterization of H2 influenza virus hemagglutinin with monoclonal antibodies: influence of receptor specificity[J]. Virologe. 1984 Oct 30; 138(2): 276-86.
[144] Yamada A, Brown LE, Webster RG. et al. Antigenic analysis of H2 influenza virus haemagglutinin with monoclonal antibodies. Vaccine. 1985 Sep; 3(3 Suppl): 195-8.
[145] Lipkind M, Weisman Y, Shihmanter E. The isolation of influenza virus from chickens in Israel and studies on its antigenic relationships with other native isolates of the same antigenicity by means of monoclonal antibodies. Comp Immunol Microbiol Infect Dis. 1987; 10(2): 133-9
[146] Wang ML, Yan JX, Lin QA. The use of micropeptide maps and monoclonal antibodies for antigenic variation analysis of influenza A viruses isolated in China from 1972 to 1983. Acta Virol. 1986 Mar; 30(2):97-102.
[147] Hovanec DL, Air GM. Antigenic structure of the hemagglutinin of influenza virus B/Hong Kong/8/73 as determined from gene sequence analysis of variants selected with monoclonal antibodies. Virology. 1984 Dec; 139(2): 384-92.
[148] Kendal AP, Phillips DJ, Webster RG. Effect of test system on the ability of monoclonal antibodies to detect antigenic drift in influenza A(H1N1) virus haemagglutinins. J Gen Virol. 1981 Jun; 54(Pt 2): 253-61.
[149] Webster RG, Laver WG, Air GM, Ward C. The mechanism of antigenic drift in influenza viruses: analysis of Hong Kong (H3N2) variants with monoclonal antibodies to the hemagglutinin molecule. Ann N Y hcad Sci. 1980; 354: 142-61.
[150] Laver WG, Air GM, Webster RG. The mechanism of antigenic drift in influenza virus: sequence changes in the haemagglutinin of variants selected with monoclonal hybridoma antibodies. Philos Trans R Soc Lond B Biol Sci. 1980 Feb 25; 288 (1029): 313-26.
[151] Gerhard W, Yewdell J, Frankel ME. Antigenic structure of influenza virus haemagglutinin defined by hybridoma antibodies. Nature. 1981 Apr 23; 290 (5808): 713-7.
[152] Ito T, Kida H, Yanagawa R. Antigenic analysis of H4 influenza virus isolates using monoclonal antibodies to defined antigenic sites on the hemagglutinin of A/Budgerigar/Hokkaido/1/77 strain. Arch Virol. 1985; 84(3-4):251-9.
[153] Vareckova E, Lyubovtseva OV, Russ G, Analysis of influenza A virus neuraminidase using lectin test and monoclonal antibodies. Acta Virol. 1986 Jul; 30(4): 281-8.
[154] Webster RG, Brown LE, Laver WG. Antigenic and biological characterization of influenza virus neuraminidase (N2) with monoclonal antibodies. Virology. 1981 Aug; 113(1): 64-72.
[155] Ueda M, Maeda A, Nakagawa N. et al. Application of subtype-specific monoclonal antibodies for rapid detection and identification of influenza h and B viruses. J Clin Microbiol. 1998 Feb; 36(2): 340-4.
[156] Capua I, Terregino C, Cattoli G, et al. Development of a DIV(differentiating infected from vaccinated animals)strategy using a vaccine containing a heterologous neuraminidase for thecontrol of avian influenza[J]. Avian eathol, 2003, 32: 47-55.
[157] 王秀荣，李冬梅，邓国华，等．AIVN_1和N_2亚型神经氨酸酶RT-PCR鉴别方法的建立[J]．中国兽医科技，2004，34(4)：9-13
[158] 徐志凯．实用单克隆抗体制备技术[J]．陕西科技出版社，1995
[159] 郑平．一种提高单克隆抗体产量的简易方法[J]．免疫学杂志，1999，3(15)：34-36
[160] 朱平．抗体试验技术[M]．吉林：长春出版社，2000
[161] 刘玉斌．动物免疫学试验技术[M]．吉林：吉林科学出版社，1989．
[162] 刘金华，史为民，吴清民，等．我国部分鸡源H9N2亚型流感病毒NS_1基因序列分析[J]．微生物学报，2003，43(5)：547-553
[163] 李建丽．H_5N_1亚型禽流感病毒非结构基因在大肠杆菌中的表达及初步应用[D]．河南农业大学硕士论文，2005，6
[164] 刘岩．1998-2002年间河南省H9N2亚型AIV的抗原性漂变研究．河南农业大学硕士学位论文，2003，6
[165] 张立春，于康震，陈化兰，等．具有HI活性的抗H5亚型流感病毒特异性单克隆抗体的研制．中国预防兽医学报，2005，5：329-332
[166] Smirnov Y A, Lipatov A S, Gitelman A D. hn epitope shared by the hemagglutinins of H1, H2, H5 and H6 subtypes of influenze A virus[J]. Acta Virol, 1999, 43(4): 237-244
[167] Shimon Perk, Ester Shimanter, Irena Gissen, et al. Involvement of Low Pathogenic H9N2 Avian Influenza as a Threat to the Poultry Industry[D]. 14th World Veterinary poultry congress final program and abstract book. 2005.
[2] B W卡尔尼克．禽病学(第十版)[M]．北京：中国农业大学出版社，1999
[3] 甘孟侯．中国禽病学[M]．北京：中国农业出版社，1999．7
[4] Oxford J S. Influenza pandemics of the 20th century with special reference to 1918: Virology, pathology and epidemiology [J]. Rev Med Virol, 2000, 10(2): 119-133
[5] 甘孟侯．全球禽流感的流行形势[J]．中国预防兽医学报，2004，26(6)：468-471
[6] Alexander D J. A reviews of avian influenza in diferent bird species [J]. Vet Microbiol, 2000, 74(1-2): 3-13
[7] 殷震，刘景华．动物病毒学(第二版)[M]．科学出版社，1997
[8] Lu X H, Cho D. Pathogenesis of and immunity to a new influenza A (H5N1) virus isolated from duck meat [J]. Avian Dis, 2003, 47(3): 1135-1140
[9] Tumpey T M. Evaluation of a hipathogenicityl15Nl avian influenza A virus isolated fromduck meat [J]. Avian Dis, 2003, 47(3): 951-953
[10] Baigent, S J, J W. McCauley. Glycosylation of hemagglution and stalk-length of neuraminidase combine to regulate the growth of avian influenza virus in tissue culture [J]. Virus Research, 2001, 79: 177-185
[11] 黄庚明，辛朝安．PCR制备地高辛标记的探针检测禽流感病毒核酸[J]．中国兽医杂志，2001，37(12)：3-7
[12] 郑腾，陈枝华．禽流感的公共卫生意义[J]．生物学通报，2002，37(4)：13-15
[13] OIE. Disease Information [J].2002, 15(29): 1-8
[14] Donateli I, Campiteli L. Characterization of H_5N_2 influenza virus from Italian poultry [J]. J Gene Virol, 2001, 82(3): 623-630
[15] 范伟兴，何海权，腾翔雁，等．近期亚洲高致病性AI流行态势简析[J]．2004，21(5)：46-48
[16] Home P J, Easterday B C. Avian influenza virus infections: Response of pheasants, ducks and geese to influenza A-turkey-Wisconsin-1966 virus [J]. Avian Dis, 1970, 14(2): 285-290
[17] 郭元吉．流感病毒生态学研究近况[J]．流行病学杂志，1980，1(1)：61-65
[18] 陈伯伦，张泽纪，陈伟斌，等．鸡A型AIV的分离与血清学初步鉴定[J]．中国兽医杂志，1994，20(10)：3-5
[19] 陈福勇，夏春．AIA／鸡／北京／1／96(H9N2)株核蛋白基因克隆和序列分析[J]．中国预防兽医学报，1999，21(2)：130-133
[20] 王永坤．鸭流感防治研究进展[J]．辽宁畜牧兽医，2003，6：32-36
[21] 席瑞珍．鸭源H_5N_1亚型AIV与鸡源H_5N_1亚型AIV的生物学特性和分子生物学特性的比较[D]．河南农业大学硕士论文，2004．6
[22] Hinshaw V. S, Bean W J, Geraci J R, et al. Characterization of two influenza A viruses from a pilot whale[J]. J. Virol, 1986, 58: 655-656
[23] Kingeborn B L, Englund R, Rott N, et al. An avian influenza A virus killing a mammalian species—the mink[J]. Arch. Virol, 1985, 86: 347-351
[24] 李雁冰，田国彬，李艳华，等．不同宿主的H_5亚型AIV对小鼠的致病性试验[A]．中国畜牧兽医学会家畜传染病学分会第十次学术研讨会禽流感研究论文专集，2003．10
[25] 焦培荣，李泽君，侯勇跃，等．四株H_5亚型AI对小鼠致病性的研究[A]．中国畜牧兽医学会家畜传染病学分会第十次学术研讨会禽流感研究论文专集，2003．10
[26] 乔传玲，于康震．AIV结构蛋白及其免疫原性[J]．预防兽医学进展，2000，2(4)：4-7
[27] 刘金华，史为民，吴清民，等．我国鸡源与人源H_9N_2亚型AI聚合酶PB_1基因关系分析[J]．中国人兽共患病杂志，2004，20(7)：589-591
[28] Yao Y, Mingay L J. Sequences in influenza virus PB_2 protein that determine productive infection for an avian influenza virus in mouse and human cell line[J]. Arch Virol, 2001, 130(12): 209-217
[29] Massin P, Vander W S. Residue 627 of PB_2 is a determinant of cold sensitivity in RNA raplicatinn of avian influenza virus in mouse and human cell line[J]. J Virol, 2001, 75(11): 5398-5404
[30] 黄庚明，辛朝安．AIV血凝素(HA)基因的研究进展[J]．广东畜牧兽医科技，2001，26(2)：3-5
[31] 李玉清，王宁．流感的分子流行病学研究进展[J]．疾病控制杂志，2001，5(1)：60-62
[32] Tonegawa K, Nobusawa E, Nakaja K, etal. Analysis of epitope recognition of antibodies induced by DNA inunization against hemagglutinin protein of influenza A virus[J]. Vaccine, 2003, 21(23): 3118-3125
[33] 吴清民．AIV核蛋白基因表达的研究[D]．中国农业大学博士论文，2002
[34] 金元昌，李景鹏，张龙，等．AIV分子生物学研究进展[J]．动物医学进展，2003，24(1)：12-15
[35] 卢建红，韦栋平，刘玉良，等．H_9N_2亚型AI核蛋白基因克隆及其序列特征[J]．中国兽医科技，2003，4：46-49
[36] 何后军，戴益民，付光华，等．AIV分子生物学进展[J]．江西农业大学学报，2004，26(2)：290-293
[37] 刘菲，郭宇飞，冉智光，等．AIV的分子生物学基础和疫苗控制研究[J]．中国家禽，2004，8(1)：195-198
[38] 刘洪雨，刘玉芬，于尔松，等．AIV的研究[J]．畜牧兽医科技信息，2005，1：4
[39] 石火英，刘秀梵．利用反向遗传技术研究H_9N_2亚型AIV传播途径的分子机制[J]．微生物学报，2006，
[40] 郭元吉，温乐英，王敏，等．我国猪群中H_9N_2亚型毒株卧和NA基因特性的研究[J]．中华试验和临床病毒学杂志，2004，18(1)：7-11
[41] 乔传玲，张建林，贾永清，等．禽流感病毒血凝素与核蛋白在重组禽痘病毒中的共表达[J]．动物医学进展，2003，24(1)：81—83
[42] 褚玲娜，詹勇，许梓荣，等．AIV的致病性和免疫性研究进展[J]．中国兽药杂志，2005，39(1)：28-32
[43] Liu W, LiH, Chen Y H. N-terminus of M2 protein could induce antibodies with inhibiitory activity against influenza virus replication[J]. FEMS lmmunol Med Microbio, 2003, 35 (2): 141—146
[44] 王传彬，孙明，赵铁柱，等．H9N2AIV分离株NS基因同源性分析[J]．预防兽医学报，2004，26(3)：177-180．
[45] 孙明，赵铁柱，王传彬，等．AIVH5N1亚型NS1基因在大肠杆菌中的表达[J]．中国试验动物学报，2004，12(4)：208-211
[46] Lamb R A, Krug R M. Orthomyxoviridae: the viruses and their replication [J]Virology, 2001, 1487-1532
[47] 郭元吉，程小雯．流行性感冒病毒及其试验技术[M]．重庆：中国三峡出版社，1997，16-20．
[48] Cassetti M C, Noah D L, Montelione G t, et al. Efficient translation of mRNAs in influenza A virus-infected cells is independent of the viral 5' untranslated region [J]. Virology, 2001, 289(2): 180-185
[49] Li Y, Yamakita Y, Krug R M, et al. Regulation of a nuclear export signal by an adjacent inhibitory sequence: The effector domain of the influenza virus NS1 protein [J]. Proc. Natl. Acad. Sci. USh, 1998, 95: 4864-4869
[50] Yuan W, AraminiJ M, Montelione G T, et al. Structural basis for ubiquitin-like 1SG15 protein binding to the NS1 protein of influenza B virus:a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS 1 protein of influenza A virus[J]. Virology, 2002, 304(2): 291-301.
[51] Chien C Y, Xu Y, Xiao R, et al. Biophysical characterization of the complex between double-stranded RNA and the N-terminal domain of the NS1 protein from influenza A virus: evidence for a novel RNh-binding mode[J]. Biochemistry, 2004,43(7): 1950-1962
[52] 李先茜，杜培革，傅桂莲，等．应用定点突变技术进行流感病毒NSI蛋白功能的研究[J]北华大学学报，2004，5(4)：316-320
[53] Skehel JJ. Polypeptidel synthesis in influenza virus-infected cells[J]. Virology, 1972, 49(1):23-36
[54] Zhirnov O P, Konakova T E, wolff T, et al. NS, protein of influenza A virus down-regulates apoptosis[J].J Virol, 2002, 76(4):1617-1625
[55] Geiss G K, Salvatore M, Tumpey T M, et al. Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: The role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza[J]. Proc Natl Acad Sci USA, 2002, 99(16): 10736-10741.
[56] Seo S H, Webster R G. Tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells [J]. J Virol, 2002, 76(3): 1071-1076.
[57] Seo S H, Hoffmann E, Webster R G, et al. Lethal H5N1 influenza viruses escape host antiviral cytokine responses [J]. Nature Med, 2002, 8(9): 950-954.
[58] Basler C F, Reid A H, Dybing J K, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes[J]. Proc. Natl. Acad. Sci. USA, 2001 27; 98(5): 2746-51
[59] TaubenbergerJ K, Reid A H, Janczewski T A, et al. Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus[J]. Philos Trans SocLond Biol Sci, 2001,356(1416):1829-39.
[60]Qiu Y, Krug R M. The influenza virus NS1 protein is a poly (A)-binding protein that inhibits nuclear export of mRNAs containing poly(A)[J]. J. Virol, 1994, 68(4):2425-2432.
[61]Chen Z, Li Y.Krug RM, et al. Influenza A virus NS1 protein targets poly(A)-binding protein of the cellular 3' end processing machinery [J]. EMBO J, 1999, 18(8) :2273 - 2283
[62]Chen ,Krug R M. Selective nuclear export of viral mRNAs in influenza virus infected cells[J]Trends Microbiol, 2000, 8:376-383.
[63]Lu Y, Qian X Y, Krug R M, et al.The influenza virus NS1 protein:a novel inhibitor of pre-mRNA splicing[J]. Genes, 1994, 8:1817-1828
[64]Fortes P, Beloso A.Ortin J, et al. Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport[J]. EMBO J, 1994,13:704-712
[65]Nemeroff M E, Barabino S M, Li Y, et al. Influenza virus NS1 protein interacts with the cellular 30 KD subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs[J].Mol Cell,1998,1:991-1000
[66]Enami K, Sato T A, Nakada S, et al. Influenza virus NS1 protein stimulates translation of the M_1 protein[J].J Virol,1994,68:1432;1437
[67]Noah D L, Tkuky, Krug R M, et al. Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS_1 A protein via its binding to a cellular protein required forthe 3' end processing of cellular pre-mRNAs[J]. Virology, 2003, 307(2):386-395.
[68]Schultz S, Dybdahl-Sissoko N, Neumann G, et al. Influenza virus NS1 protein induces apoptosis in cultured cells[J].J Virol, 2001, 75(17):7875-7881
[69]Takizawa, Lowy R J. Influenza virus induction of apoptosis by intrinsic and extrinsic mechanisms[J]. Int Rev Immunol, 2003, 22(5-6):425-49
[70]Zhirnov O P Konakova T E, wolff T, et al. NS_1 protein of influenza A virus down regulates apoptosis[J].J Virol, 2002, 76(4):1617-1625.
[71]Lu Y, Wambach, Katze M G, et al. Binding of the influenza virus NS1 protein to double stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor[J]. Virology, 1995 , 214 (1):222-228
[72]Garcia-Sastre A, Egorov D, Matassov, et al. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems[J]. Virology, 1998, 252(2):324-330
[73]Talon J, Salvatore M, Neill R E, et al. Influenza A and B viruses expressing altered NS1 proteins:a vaccine approach[J]. Nat1 Acad Sci USA, 2000, 97(8):4309-4314
[74]Stasakova J, Ferko B, Kittel C, et al. Influenza A mutant viruses with altered NS1 protein function provoke caspase-1 activation in primary human macrophages, resulting in fast apoptosis and release of high levels of interleukins beta [J]. J Gen Virol, 2005, 86(Pt1): 185-195.
[75] Ferko B, Stasakova J, Romanova J, et al. Immunogenicity and protection efficacy of replication-deficient influenza A viruses with altered NS1 genes[J]. J Virol, 2004, 78(23): 13037-13045
[76] Wang X M, Li, H, Zheng, et al. Influenza A virus NS1 protein prevents the activation of NF-κB and induction of type I IFN[J]. J. Virol, 2000, 74(24): 11566-11573
[77] Kazufumi S, Kazumichi K. Expression of host genes in influenza virus infected cells [J]. Uirusu., 2004, 54(2): 189-96
[78] Zurcher Y, Marion R M,Ortin J, et al. Protein synthesis shut-off induced by influenza virus infection is independent of PKR aetivity[J]. J Virol, 2000, 74: 8781-8784
[79] Bergmann M, Garcia-Sastre A. Influenza virus NS1 protein counteracts PKR-mediated inhibition of replieation[J]J. Virol, 2000, 74(13): 6203-6206
[80] Stephan, Ludwig, Xiuyan, et al. Yhe Influenza A Virus NS1 Protein Inhibits Activation of Jun N-Terminal Kinase and AP-1 Transcription Faetors[J]. Journal of Virology, 2002, 76(21): 11166-11171
[81] Geiss G K, Salvatore M, Tumpey T M, et al. Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: The role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza[J]. Proc Natl Acad Sci USA, 2002, 99(16): 10736-10741
[82] Bergmann I E, Mello P A, Neitzert E, et al. Diagnosis of persistent aphthovirus infection and its differentiation from vaccination response in cattle by use of enzyme linked immunoelectrotransfer blot analysis with bioengineered nonstructural viral antigens[J].J Vet Res, 1993, 54(6): 825-831.
[83] Rodriguez A, Dopazo J, Saiz J C, et al. Immunogenicity of non-structural proteins of foot-and-mouth disease virus: differences between infected and vaccinated swine[J]. ArchVirol, 1994, 136: 123-131
[84] Diego D M, Brocchi E, Mackay D, et al. The non-structural polyprotein 3ABC of foot-and-mouth virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle[J]. ArchVirol, 1997, 142: 2021-2033.
[85] Malirat V, Neitzert E, Bergmann I E, et al. Detection of cattle exposed to foot-and-mouth disease virus by means of an indirect ELISA test using bioengineered nonstructural polyprotein 3ABC[J]. Vet Q, 1998, 20(2): 24-26.
[86] Bergmann I E, Malirat E V, Neitzert E, etal. Improvement of a serodiagnostic strategy for foot-and-mouth disease virus surveillance in cattle under systematic vaccination: a combined system of an indirect ELISA-3ABC with anenzyme-linked immunoelectrotransfer blot assay [J]. ArchVirol, 2000, 145: 473-489.
[87] 王宏伟，孙明，田克恭，等．口蹄疫病毒3ABC、3AB、3BC基因克隆、表达及其抗体消长规律的研究[J]．中国农业科学，2004，37(7)：1065-1070
[88] 朱彩珠，尤永进，葛艳，等．用3AB-ELISA鉴别猪口蹄疫病毒感染与疫苗免疫猪的研究[J]．中国兽医科技，2005，35(1)：14-16
[89] 罗宝正，薄清如，王伟毅，等．口蹄疫病毒非结构蛋白3ABC基因的克隆与原核表达．农业生物技术学报，2005，13(2)：167—170
[90] 罗宝正，薄清如，陈金项，等．昆虫杆状病毒系统表达口蹄疫病毒3ABC基因．中国病毒学，2005，20(3)：303—306
[91] Silberstein E, Kaplan G, Taboga O, etal. Foot—and—mouth disease virus-infected but not vaccinated cattle develop antibodies against recombinant 3AB nonstructuralProtein [J]. Arch Virol, 1997, 142: 795—805
[92] 吕建强，陈焕春，赵俊龙，等．猪细小病毒NS1基因的克隆及表达[J]．中国兽医学报2003，23(5)：435-437
[93] 王君伟，等．鹅细小病毒NSI基因的克隆及原核表达．中国兽医杂志，2004，40(1)：10-13
[94] 杨敬，薛小平，雷迎峰，等．丙型肝炎病毒ns区全长基因在大肠杆菌中的高效表达及纯化．生物技术通讯，2005，1：13-15
[95] 李卫华，赵连三，周陶友，等．表达丙型肝炎病毒NS3抗原的重组腺病毒构建及体外表达．中国病毒学，2005，20(2)：101—104
[96] 沈成利，王弋嘉，石英，等．SARS冠状病毒多聚酶区抗原在感染诊断中的作用探讨．解放军医学杂志，2005，30(8)：720-724
[97] 张大鹏，王进，华子春．SARS病毒蛋白保守性分析．东南大学学报(医学版)，2005，24(1)：32—35
[98] Wang SJ, Boyle RH, Dearest VL, et al. Immunassay targeting nonstructural Protein 5 to differentiate West Nile virus infection from dengue and St. Louis encephalitis virus infections and from fiavivirus vaccination[J]. CLin Mcrobiol, 2003, 41(9): 4217
[99] Gzaki H, Sugiura T, Sugita S etal. Detection of antibodies to the nonstructural protein(NSI)of influenza A virus allows distinction between vaccinated and infected houses[J]. Veterinary Mcrobiolo gy, 2001, 82(2): 111—119.
[100] Birch-Machin I, Rowan A, Pick J, et al. Expression of the nonstructural protein NS1 of equine influenza A virus: detection of anti-NS1 antibody in post infection equine sera[J]. J. Virol Methods, 1997, 65: 255-263.
[101] Ozaki H, Sugiura T, Sugita S, et al. Detection of antibodies to the nonstructural protein(NS1)of influenza A virus allows distinction between vaccinated and infected horses[J]. Vet Microbiol, 2001, 82(2): 111-119
[102] 杨涛，刘明，张云，等．H_5N_1亚型高致病性AIVNS基因的克隆及序列分析[J]．中国兽医科技，2005，35(1)：5-9
[103] 张志珍，洪文珊，李康生，等．流感病毒NS1基因的克隆及原核表达载体的构建[J]．中国病毒学，2004，19(5)：449-453
[104] Tumpey T M, Alvarez R, Swayne D E, et al. Diagnostic approach for differentiating infected from vaccinated poultry on the basis of antibodies to NS1, the nonstructural protein of influenza A virus[J]. J Clin Microbiol, 2005, 43(2): 676-83.
[105] 王泽霖，李建丽，陈少渠，等．非结构蛋白NS1-ELISA检测禽流感野毒感染抗体[J]．科技导报，2006，3
[106] Kurtz J, Manvell R J, Banks J, et al. Avian influenza virus isolated from a woman with conjunctivitis[J]. Lancet, 1996, 348: 901-902.
[107] 王海燕，刘文博，刘秀梵，等．H9N2亚型A型流感[J]．中国禽业导刊，2004，21(9)：30-31
[108] 刘红旗，黄勇，程坚，等．在疫苗免疫选择压力下H9N2亚型禽流行性感冒病毒HA基因的遗传变异[J]．病毒学报，2002，18(2)：149
[109] 郭元吉，谢健屏，吴昆昱，等．流感病毒广州／333／99(H9N2)毒株基因组特性的研究[J]．中华试验和临床病毒学杂志，2002，16(2)：142
[110] Katz J M, Lu X. Pathogenesis of and immunity to avian influenza A H_5 viruses[J]. Biomed Pharmacother, 2000, 54(4): 178-187
[111] Swayne D E, Suarez D L. Highly pathogenic avian influenza virus[J]. Rev Sci Tech, 2000, 19(2): 463-82
[112] Rott R, Bosch F X, Garten, et al. The pathogenic determinant of influenza virus[J]. Veterinary Microbiology, 1992, 33: 303-310.
[113] Ogawa. T Genes involved in the virulence of avian influenza virus [J]. Virology, 1981, 113: 304-313
[114] Horimoto T, Kawaoka Y. Reverse genetics provides direct evidence for a correlation of hem agglutinin cleavability and virulence of an avian influenza A virus[J]. J. Virol, 1994, 68(5): 3120-3128
[115] Subbarao K, Chen H, Swayne D, et al. Evaluation of a genetically modified reassortant H_5N_1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics[J]. Virology, 2003, 305: 192-200
[116] Michael L, Perdue. How can a virus suddenly become very pathogenic?[J]. World poultry elsevier special, 2000, 6: 9-10
[117] Senne D A, Panigraphy R G, Webster, et al. Survey of the hemagglutinin (HA)cleavagesite as a marker of pathogenicity potential[J]. Avian Disease, 1996, 40(1): 425-437
[118] Besphanda K L, Fried V. A, Webste R. G, et al. Glycosylation affects cleavage of avian influenza virus hemagglutinin and regulates virulence[J]. J Virol, 2000, 74(3): 1443-1450
[119] Matrosovich M, Zhou N, Kawaoka Y, et al. The surface glycoproteins of H_5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties[J]. J Virol, 2003, 73(2): 1146-55.
[120] Goto H, Kawaoka Y. A novel mechanism for the acquisition of virulence by a human influenza A virus[J]Proc. Natl. Acad. Sci. USA, 1998, 95: 10224-10228
[121] Castrucci M R, Kawaoka Y. Biologic importance of neuraminidase stalk length in influenza A virus[J]. J. Virol, 1993, 67(2): 759-764
[122] Hatta M, Gao P, Halfmann P, et al. Molecular basis for high virulence of Hong Kong H_5N_1 influenza A virus[J]. Science, 2001, 293: 1840-1842
[123] Subbarao E K, London W, Mupphy B R, et al. A single amino acid in the PB_2 gene of influenza A virus is a detexm inant of host range[J]. J. Virol, 1993, 67(4): 1761-1764
[124] Chen W, Calvol P A, Malide D, et al. A novel influenza A virus mitochondrial protein that induces cell death[J]Nature Medicine, 2001,7(12): 1306-1312.
[125] Wang X, Castro A E, Castro M D, et al. Production and evaluation criteria of monoclonal antibodies to the hemagglutinin of the H7N2 subtype of avian influenza virus[J]. J Vet Diagn Invest, 2000, 12(6): 503
[126] Olsencw. Virologic and serologic surveillance for human, swine and avian influenza virus infections among pigs in the north-central United States[J]. Arch Virol, 2000, 145(7): 399-419
[127] Anirnov Iua. A common antigenic epitope in influenza A virus(H1,H2, H5, H6) Hemagglutinin [J].Vopr Viruol, 1999, 44(3): 11-15
[128] Fukushi H. Host species-related antigenic groups of avian influenza viruses possessing H4 hemagglutinin revealed by monoclonal antibodies[J]. Arch Virol, 1982, 72(93): 17-21
[129] 邵红霞，秦爱建，钱琨等．抗禽流感病毒H5亚型血凝素特异性单克隆抗体的研制[J]．中国动物检疫，2002．19(8)
[130] 秦爱建，邵红霞，钱琨，等．抗禽流感病毒和H5和H9亚型血凝素特异性单克隆抗体的研制及应用[J]．中国预防兽医学报，2003，25(3)：161
[131] 王泽霖，黄娟，马仲彬，等．斑点免疫金渗滤法快速检测禽流感病毒[A]．中国畜牧兽医学会禽病学分会第十二次学术讨论会论文集，2004，3：87-90
[132] 赵维波，陈美才，刘忠华等．抗禽流感病毒H5N1亚型单克隆抗体制备[J]．中国试验动物学报，2004，12(4)
[133] 曾艳兵，焦新安，潘志明，等．抗H9亚型禽流感病毒血凝素单克隆抗体的制备及初步鉴定[J]．细胞与分子免疫学杂志，2004．20 C6)：702～704
[134] 王海燕，刘文博，刘秀梵，等．H9亚型禽流感病毒分型单克隆杭体的研制及初步应用．中国家禽，2004，26(10)：21-27
[135] 王传彬，田新生，曹振，等．H5亚型禽流感病毒单抗-生物素捕获ELIsA的建立．中国试验动物学报，2004，12(4)：204-207
[136] 孔晶，吴艳涛，刘文博，等．禽流感病毒单克隆抗体的研制及初步应用．中国动物检疫，2004，21(10)：20-22
[137] 龙进学，郝贵杰，唐应华，等．H5N1亚型禽流感病毒血凝素单克隆抗体的制备及其应用．中国兽医科技，2005，35(6)：450—454
[138] 马仲彬．抗H9N2亚型禽流感病毒单克隆抗体杂交瘤细胞株的建立及快速检测试纸条的研制．河南农业大学硕士论文，2005，6
[139] 马秀丽，宋敏训，黄兵，等．H5亚型禽流感单克隆抗体的研制及应用．家禽科学，2005， 6：11-13
[140] 曹振．禽流感病毒H5亚型血凝素单克隆抗体的制备及捕获ELISA方法的建立[D]．中国农业大学硕士学位论文，2005，6
[141] 王凯，金宁一，鲁会军等．禽流感病毒H7亚型血凝素单克隆抗体的制备[J]．中国预防兽医学报，2005年，27(6)：456～457
[142] Zakstel'skaia LIa, Isachenko VA, Oskerko TA. Changes in the antigenic composition of virus A (H1N1) hemagglutinin in 1977-1982 detected by using monoclonal antibodies. Yopr Virusol. 1984 Jan-Feb; 29(1): 62-6.
[143] Yamada A, Brown LE, Webster RG. Characterization of H2 influenza virus hemagglutinin with monoclonal antibodies: influence of receptor specificity[J]. Virologe. 1984 Oct 30; 138(2): 276-86.
[144] Yamada A, Brown LE, Webster RG. et al. Antigenic analysis of H2 influenza virus haemagglutinin with monoclonal antibodies. Vaccine. 1985 Sep; 3(3 Suppl): 195-8.
[145] Lipkind M, Weisman Y, Shihmanter E. The isolation of influenza virus from chickens in Israel and studies on its antigenic relationships with other native isolates of the same antigenicity by means of monoclonal antibodies. Comp Immunol Microbiol Infect Dis. 1987; 10(2): 133-9
[146] Wang ML, Yan JX, Lin QA. The use of micropeptide maps and monoclonal antibodies for antigenic variation analysis of influenza A viruses isolated in China from 1972 to 1983. Acta Virol. 1986 Mar; 30(2):97-102.
[147] Hovanec DL, Air GM. Antigenic structure of the hemagglutinin of influenza virus B/Hong Kong/8/73 as determined from gene sequence analysis of variants selected with monoclonal antibodies. Virology. 1984 Dec; 139(2): 384-92.
[148] Kendal AP, Phillips DJ, Webster RG. Effect of test system on the ability of monoclonal antibodies to detect antigenic drift in influenza A(H1N1) virus haemagglutinins. J Gen Virol. 1981 Jun; 54(Pt 2): 253-61.
[149] Webster RG, Laver WG, Air GM, Ward C. The mechanism of antigenic drift in influenza viruses: analysis of Hong Kong (H3N2) variants with monoclonal antibodies to the hemagglutinin molecule. Ann N Y hcad Sci. 1980; 354: 142-61.
[150] Laver WG, Air GM, Webster RG. The mechanism of antigenic drift in influenza virus: sequence changes in the haemagglutinin of variants selected with monoclonal hybridoma antibodies. Philos Trans R Soc Lond B Biol Sci. 1980 Feb 25; 288 (1029): 313-26.
[151] Gerhard W, Yewdell J, Frankel ME. Antigenic structure of influenza virus haemagglutinin defined by hybridoma antibodies. Nature. 1981 Apr 23; 290 (5808): 713-7.
[152] Ito T, Kida H, Yanagawa R. Antigenic analysis of H4 influenza virus isolates using monoclonal antibodies to defined antigenic sites on the hemagglutinin of A/Budgerigar/Hokkaido/1/77 strain. Arch Virol. 1985; 84(3-4):251-9.
[153] Vareckova E, Lyubovtseva OV, Russ G, Analysis of influenza A virus neuraminidase using lectin test and monoclonal antibodies. Acta Virol. 1986 Jul; 30(4): 281-8.
[154] Webster RG, Brown LE, Laver WG. Antigenic and biological characterization of influenza virus neuraminidase (N2) with monoclonal antibodies. Virology. 1981 Aug; 113(1): 64-72.
[155] Ueda M, Maeda A, Nakagawa N. et al. Application of subtype-specific monoclonal antibodies for rapid detection and identification of influenza h and B viruses. J Clin Microbiol. 1998 Feb; 36(2): 340-4.
[156] Capua I, Terregino C, Cattoli G, et al. Development of a DIV(differentiating infected from vaccinated animals)strategy using a vaccine containing a heterologous neuraminidase for thecontrol of avian influenza[J]. Avian eathol, 2003, 32: 47-55.
[157] 王秀荣，李冬梅，邓国华，等．AIVN_1和N_2亚型神经氨酸酶RT-PCR鉴别方法的建立[J]．中国兽医科技，2004，34(4)：9-13
[158] 徐志凯．实用单克隆抗体制备技术[J]．陕西科技出版社，1995
[159] 郑平．一种提高单克隆抗体产量的简易方法[J]．免疫学杂志，1999，3(15)：34-36
[160] 朱平．抗体试验技术[M]．吉林：长春出版社，2000
[161] 刘玉斌．动物免疫学试验技术[M]．吉林：吉林科学出版社，1989．
[162] 刘金华，史为民，吴清民，等．我国部分鸡源H9N2亚型流感病毒NS_1基因序列分析[J]．微生物学报，2003，43(5)：547-553
[163] 李建丽．H_5N_1亚型禽流感病毒非结构基因在大肠杆菌中的表达及初步应用[D]．河南农业大学硕士论文，2005，6
[164] 刘岩．1998-2002年间河南省H9N2亚型AIV的抗原性漂变研究．河南农业大学硕士学位论文，2003，6
[165] 张立春，于康震，陈化兰，等．具有HI活性的抗H5亚型流感病毒特异性单克隆抗体的研制．中国预防兽医学报，2005，5：329-332
[166] Smirnov Y A, Lipatov A S, Gitelman A D. hn epitope shared by the hemagglutinins of H1, H2, H5 and H6 subtypes of influenze A virus[J]. Acta Virol, 1999, 43(4): 237-244
[167] Shimon Perk, Ester Shimanter, Irena Gissen, et al. Involvement of Low Pathogenic H9N2 Avian Influenza as a Threat to the Poultry Industry[D]. 14th World Veterinary poultry congress final program and abstract book. 2005.