猪瘟病毒E2囊膜糖蛋白B-cell线性化表位的表达及免疫活性研究
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摘要
本实验利用含猪瘟病毒石门株(CSFV Shimen strain)全基因的质粒为模板,扩增囊膜糖蛋白E2中B/C抗原区和A╱D抗原区的线性化B-Cell表位。扩增了两个单独的基因片段(E2蛋白A╱D区和B/C区基因序列)与一个以上两基因片段融合的基因片段(E2蛋白A/D区连接B/C区基因序列),将扩增的三个片段分别插入原核表达载体pET-32a(+)中,构建成重组表达质粒pTA,pTB和pTC。将构建成功的阳性质粒转化原核表达菌BL21(DE3),对转化重组菌用IPTG进行诱导表达并对诱导表达条件进行优化,使目的蛋白得到高效表达。
应用Ni-NTA亲和层析柱纯化三种重组蛋白,纯化产物经SDS-PAGE电泳鉴定其纯化效果,并用Western-blot检验重组蛋白对猪瘟阳性血清特异的反应原性。
在此基础上,将三种纯化的蛋白定量后等体积混合弗氏佐剂免疫猪。经过两次免疫后检测免疫动物的猪瘟特异性抗体消长和免疫相关细胞因子变化情况。
结果表明,三种蛋白均可刺激机体产生体液免疫应答和细胞免疫应答,pTC蛋白的免疫效果明显优于另两种蛋白,并且非常接近商品化细胞疫苗的免疫效果,pTB和pTC刺激机体产生的猪瘟抗体均达到阳性。三种重组蛋白诱导的细胞因子应答符合免疫应答规律。说明三种重组蛋白具有良好的免疫原性和免疫效果。
Three recombinant prokaryotic vectors, pTA, pTB and pTC were constructed containing classical swine fever virus C strain E2 linear B-cell epitope gene(A/D antigenic domain and B/C antigenic domain) using pET 32a(+) expressing vectors. Their expressing characteristics were compared and it was found that pTA, pTB and pTC in BL21(DE3) can present high production of recombinant protein. Then, expression of protein were optimized by inducing in different time and different concentration of IPTG. After that, the recombinant protein was purified by Ni-NTA affinity chromatography. Purified protein was quantified and detected by SDS-PAGE and Western-blot. The purified proteins mixing with Freud's adjust and inoculated to porcines. After vaccination, specificity antibody titers against CSFV and relative cytokines production were detected by ELISA.
应用Ni-NTA亲和层析柱纯化三种重组蛋白,纯化产物经SDS-PAGE电泳鉴定其纯化效果,并用Western-blot检验重组蛋白对猪瘟阳性血清特异的反应原性。
在此基础上,将三种纯化的蛋白定量后等体积混合弗氏佐剂免疫猪。经过两次免疫后检测免疫动物的猪瘟特异性抗体消长和免疫相关细胞因子变化情况。
结果表明,三种蛋白均可刺激机体产生体液免疫应答和细胞免疫应答,pTC蛋白的免疫效果明显优于另两种蛋白,并且非常接近商品化细胞疫苗的免疫效果,pTB和pTC刺激机体产生的猪瘟抗体均达到阳性。三种重组蛋白诱导的细胞因子应答符合免疫应答规律。说明三种重组蛋白具有良好的免疫原性和免疫效果。
Three recombinant prokaryotic vectors, pTA, pTB and pTC were constructed containing classical swine fever virus C strain E2 linear B-cell epitope gene(A/D antigenic domain and B/C antigenic domain) using pET 32a(+) expressing vectors. Their expressing characteristics were compared and it was found that pTA, pTB and pTC in BL21(DE3) can present high production of recombinant protein. Then, expression of protein were optimized by inducing in different time and different concentration of IPTG. After that, the recombinant protein was purified by Ni-NTA affinity chromatography. Purified protein was quantified and detected by SDS-PAGE and Western-blot. The purified proteins mixing with Freud's adjust and inoculated to porcines. After vaccination, specificity antibody titers against CSFV and relative cytokines production were detected by ELISA.
引文
[1]蔡宝祥.家畜传染病学,第四版[M].北京:中国农业出版社,2001,147-153,
[2]殷震,刘景华.动物病毒学,第三版[M].北京:科学出版社,1997,652-664.
[3]F.A.Murphy,C.M.Fauquet,D.H.L.Dishop,S.S.Ghabrial,A.W.Jarvis,P.Martelli,M.A.Mayo and M.D.Summers,Virus taxonomy.Sixth report of the international committee on taxonomy of viruses[J].Arch.Virol.Supp 1.10,1995,415-427.
[4]Deng R,Brock K.5'and 3'untranslated regions of pestivirus genome:primary and secondary structure analyses[J].Nucleic Acid Res.1993,21:1949-1957.
[5]Kozak M.The scanning model for translation:an update[J],J.Cell.Biol.1989,108:229-241
[6]肖明,张楚瑜,祝志展等猪瘟病毒基因组非编码区的定性、定量与结构分析[J].科学通报,2001,46:544-550.
[7]Stark,R,Rumenapf T,Meyers G,et al.Processing of pestivirus:cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus[J].J.Virol.1993,67:7088-7099.
[8]Thiel H.J,Stark R,Weiland E,et al.Hog cholera virus:molecular composition of irions from a pestvirus[J].J.Virol.1991,65:4705-4712.
[9]ELBER K,Tautz P,Becher D,et al.Processing in the pestivirus E2-NS2region:identification of protein p7 and E2p7[J].J.Virol.,70:4131-4135.
[10]Rumenapf T,Ungher C,Strauss J.H.H,et al.Processing of the envelope glycoproteins of pestivirus[J].J.Virol.1993,67:3288-3294.
[11]Elbers K,Tautz N,Becher P,et al.Processing in the pestivirus E2-p7 region:identification of protein p7 and E2p7[J].J.Virol.1996,70:4131-4135.
[12]Wensvoort GBoonstra J,Bodzinga B G.Immunoaffinity purification and characterization of the envelope protein E1 of hog cholera virus[J].J.Gen.Viro 1.1990,71:531-540.
[13]Stark R,Rumenapf T,G Meyers,et al.Genomic localization of hog cholera virus glyvoproteins[J].Virology,1990,174:286-289.
[14]Wiskerchen M M,Collett M S.Pestivirus gene expression:p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein[J].Virology.1997,165:191-199
[15]Hust M M,Himes G,Newbigin E,et al.Glycoprotein E2 of the classical swine fever virus:expression in insect cells and identification as aribonuclease[J].Virology,1994,200:588-565.
[16]Wendisch J N,S chncider R,Stark R.et al.RNase of classical swine fever virus:biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies[J].J.Virol.1996,70:352-358.
[17]韩雪清,刘湘涛,赵启祖.猪瘟病毒遗传发生关系分析[J].中国兽医科技,1999,29:3-7.
[18]王宁,付烈振,张楚瑜.猪瘟病毒囊膜糖蛋白E0的RNA酶活性及其研究进展[J].微生物学通报,1998,25:304-300.
[19]Bruschke C H 19,Hust M M,Moormann R J,et al.Glycoprotein E~(rns) of pestivirus induces apoptosis in lymphocytes of several species[J].J.Virol.1997, 71:6692-6696.
[20]Hust M M,Pauoto F E,Hoekman A,et al.nactivation of the RNase activity of glycoprotein E~(rns) of classical swine fever virus results in a cytopathogenic virus[J].J.Virol.1998,729:151-157
[21]Wendisch J M,Schncider R,Stark R,et al.RNase of classical swine fever virus:biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies[J].J.Virol.1996,70:302-308.
[22]Van Rijn PA,van Gennip H G P,Mei jier E J,et al.Epitople mapping of envelope glycoprotein E1 of hog cholera virus strain Brescia[J].J Gen.Virol.1993,74:2053-2060.
[23]Min L,Fang L,Mallery M,et al.Deletioin of structural glycoprotein E2 of classical swine fever virus strain Alfort/187 resolve a linear epitopes of monoclonal antibodies WH303 and N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum[J].J.Virol.2000,11619-11625.
[24]Yu M,Wang L F,Shiell B T,et al.Fine mapping of a C-terminal linear epitope highly conserved among the major envelope glycoprotein E2(gp51 to gp54) of different pestivirusl[J].Virololgy.1996,222:289-292.
[25]Hust M M,Moormann R J.Inhibition of pestivirus infection in cell culture by envelope proteins E~(rns) and E2 of classical swine fever virus:E~(rns) and E2 interact with different receptors[J].J Gen.Virol.1997,78:2779-2787.
[26]Collier A J,Gallego J,Klinck R,et al.A conserved RNA structure within the HCV IRES eIF3-bingding site[J].Nat.Struct.Biol.2002,9:375-380.
[27]Lowings P,Ibata G,Needham,et al.Classical swine fever virus diversity and evolution[J].J Gen.Virol.1996,77:1131-1132.
[28]赵坛,王在时,王琴等.猪瘟病毒石门株不同代次E2基因主要抗原编码区序列差异分析[J].中国兽医杂志,2002,38:7-10.
[29]付烈振,张楚瑜等.猪瘟病毒石门株E2基因序列分析[J].中国农业科学,1999,32:74-78.
[30]卞琴,李博,王在时.猪瘟病毒强弱毒株和野毒株E2全基因序列测定及比较分析[J].微生物学报,2001,41:319-328.
[31]韩雪清,李红卫,刘湘涛等.中国猪瘟兔化弱毒株(C株)兔脾组织毒土要保护性抗原E2(gp55)基因序列分析[J].畜牧兽医学报,2001,32:52-57.
[32]李红卫,涂长春,吕宗吉等.异源猪瘟病毒C株E2基因保护性抗原编码区的序列分析与比较[J].畜牧兽医学报,1998,18:112-114.
[33]李红卫,刘湘涛,李小兵.我国猪瘟病毒兔化弱毒株囊膜糖蛋白E0基因的克隆及序列测定[J].中国病毒学,1999,14:169-173.
[34]Xu J,Mendez E,Caron P R,et al.Bovine viral diarrhea virus NS3 serine protease:polyprotein cleavage sites and molecular model of an enzyme essential for pestivus replication[J]J.Virol,1997,71:5312-5322
[35]Loving CL,Brockmeier SL,Sacco RE,et al.Differential type Ⅰ interferon activation and susceptibility of dendritic cell populations to porcine arterivirus [J].Immunology,2006,76(23):154-58.
[36]Kaden V,Hubert P,Strebelow G,et al.Comparison of laboratory diagnostic methods for the detection of infection with the virus of classical swine fever in the early inspection phase:an experimental study[J].Berl.Munch.Tierarztl.Wochenschr.1999,112:52-57.
[37]Diaz de A,Sunez J I,Ganges L,et al.An RT-PCR assay for the specific detection of classical swine fever virus in clinical samples[J].Vet.Res.1998,29:431-440.
[38]Vydelingum S,Tao T,Balazse K,et al.Comparison of a reverse transcription-polymerase chain reaction assay and virus isolation for the detection of classical swine fever virus[J].Rev.Sci.Tech.1998,17:674-681.
[39]Meng Yu et al.Fine mapping of a C-terminal linear epitope highly conserved among the major envelope glycoprotein E2(gp51 to gp54) of different pestiviruses[J].Virology,1996,222:289-292.
[41]Lin M et al.Deletions of structural glycoprotein E2 of classical swine fever virus strain Alfort/187 resolve a linear epitope of monoclonal antibody WH303 and the minimal N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum[J].J Virol 2000;74(24):11619-25.
[42]G.R.Risatti et al.Identification of a novel virulence determinant within the E2structural glycoprotein of classical swine fever virus[J].Virology,2006,355:94-101.
[43]张富强,张念祖.应用噬菌体展示技术进行猪瘟病毒结构糖蛋白E2和E~(rns)中和抗原表位鉴定和比较[M].中国人民解放军军需大学博士论文,2002
[44]肖昌,余兴龙,涂长春等.利用随机肽库鉴定猪瘟病毒E2蛋白抗原表位[J].中国兽医学报,2005,25(5):449-452
[45]Ying Hua Chen et al.Candidate peptide vaccine induced protection against classical swine fever virus[J].Vaccine,2002,21:167-173
[46]Ying Hua Chen et al.Candidate multi-peptide-vaccine against classical swine fever virus induced potent immunity with serological marker[J].Vaccine,2005,23:3630-3633
[47]Ying Hua Chen et al.Spying the neutralizing epitopes on E2 N-terminal by candidate epitope-vaccines against classical swine fever virus[J].Vaccine,2006,24:4029-4034
[48]Ying Hua Chen et al.Candidate peptide-vaccine induced potent protection against CSFV and identified a principal sequential neutralizing determinant on E2[J].Vaccine,2006,24:426-434
[49]Ying Hua Chen et al.Candidate peptide-vaccines induced immunity against CSFV and identified sequential neutralizing determinants in antigenic domain A of glycoprotein E2[J].Vaccine,2006,24:1906-1913
[49]Rumenupf T,Stark R,Meyers G,et al.Structual proteins of hoglera virus expressed by vaccine virus further characterization and induction of protective immunity[J].Virol,1991,65:589-597.
[50]Hahn J.Construction of recombinant swine poxviruses and expression of classical swine fever virus E2 protein[J].Virol Methods,2001,93(1-2):49-56
[51]Berns.A live attenuated pseudorabies virus expressing envelop glycoprotein El of hog cholera virus protects swine against both the pseudorabies and hog cholera [J].Virol,1991,65:2761-2765.
[52]Van Gennip H G P,Van Rijin PA,Widojoatmodjo M 1,et al.Chimeric classical swine fever viruses containing envelope protein E~(rns) or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response[J]. Vaccine, 2001, 19:447-4}9.
[53] De Smit A J,Bouma A, Van Gennip H G P, et al.Chimeric(marker)C-stain virus induce classical protection against virulent classical swine fever virus vaccinated pigs[J].Vaccine, 2001, 19:1467-1476.
[54] Hust M M,Westra D F,Wenvoort G, et al. Glycoprotein E2 of hog cholera virus expressed in insect cell protects swine fran hog cholera[J].Virol, 1993, 67:5435-5442.
[55] Moormann R J,Bouma A. Development of classical swine fever subunit marker vaccine and companion diagnostic test[J].Vet Microbiol,2000,73(2-3):209-219.
[56] Bouma A, De Smit A J, De Jong M C, et al. Determination of the onset of the herd- immunity induced by E2 subunit vaccine against classical swine fever virus[J].Vaccine, 2000, 18(14):1374-1381.
[57] De Smith A J, Van Gennip H G P,Miedema G K W, et al. Prevention of transplacental transmission of moderate-virulent classical swine fever virus (CSFV) after as single or double vaccination with a CSFV E2 subunit vaccine [J].Vet Quart, 2000,22:150-153.
[58] Deevlf J, Laevens H, Koenen K, et al. An experimental infection with classical swine fever virus in F2 subunit marker vaccinated and in non vaccinated pigs[J]. Vaccine, 2001, 19:75-482.
[59] An S H, Song ,1 K, Kim B H, et al. Molecular biological approach for the eradication of classical swine fever in Korea[A].OIE symposium on classical swine fever (hog Cholera) C;.Birminghan, UK, 1998. 919.
[60] Yu X,Tu C,Li H, et al. DNA-mediated protection against classical swine fever virus [J].Vaccine, 2001, 19:1520-1520
[61] A. D. Guise, S. M. West and J. B. Chaudhuri. Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies[M].Mol. Biotech. 6(1996), 53-64.
[62] B. E. Fischer. Renaturation of recombinant proteins produced as inclusion bodies[J] Biotech .adv. 12(1994), 89-101.
[63] Steven B f, Bergstrom C C, Loughin T A, et al. Development of a recombination nucleoprotein-based enzyme-linked immumosorbent assay for quantification of antibodie against porcine reproductive and respiratory virus. Clin Diagn Lab Immumol. 2000. 7(4): 700-702
[64]王川庆,罗俊,丁利民,等．囊素在猪体的应用效果研究[J].中国兽医杂志,2002,38(4): 12,13
[65] D. J. Rowlands, D. V. Sangar, P. Talbot, F. Brown. Furrther evidence for multiple proteins in the foot-and-mouth disease virus particle[J].J Gen. Virol 13 (1971), 3-84.
[66] M. P. Broekhui jsen, T. Blom, M. Kottenhagen, P. H. Pounel, R. H. Meloen, S. J. Barteling, B. E. Engel Valk. Synthesis of fusion protein containing antigenic determinants of foot-and-mouth disease virus[J].Vaccine. 4 (1986), 119-124.
[67] A. M. Haberman, C. Moller, D. McGreedy and W. V. Gerhard. A large degree of functional diversity exists among helper T cells specific for the same antigenic site of influenza hemagglutinin[J].J. Immunol. 145, 1990, 3087-3094.
[68] H. Zaghouani, M. Krystal, H. Kuzu, T. Moran, H. Shah, Y. Kuzu, J. Schulman and C. Bona. Cells expressing an H chain Ig gene carrying a viral T cell epitope are lysed by specific cytotoxic Tcells[J].J. Immunol. 1489, 1992, 3604-3649.
[69] A. C. Palmenberg. Sequence alignments of picornaviral capsid proteins. In: B. L. Semler and E. Ehrenfeld, Editors, Molecular aspects of picornavirus infection and detection[J].American Society for Microbiology, Washington D C, 1989, 211-241.
[70] H. Zaghouani, S. A. Anderson, K. E. Sperber, C. Daian, R. C. Kennedy, L. Mayer and C. A. Bona. Induction of antibodies to the human immunodeficiency virus type I by immunization of baboons with immunoglobulin molecules carrying the principal neutralizing determinant of the envelope protein[J]. Proc. Net. Acad. Sci, 1995, 631-635
[71] E. W. C. Chan, H. T. along, S. C. S. Cheng .An immunoglobulin G based chimeric protein induced foot-and-mouth desease specific immune response in swine[J].Vaccine. 19 , 2000, 038-046.
[72] M. D. Winther, G. Allen, R. H. Bomford and F. Brown. Bacterially expressed antigenic peptide from foot-and-mouth disease virus capsid elicits variable immunologic responses in animals[J].J Immunol. 156, 1986, 1835-1840.
[73] Z. X. Zheng, Q. X. Xu, C. Q. Mao, H. X. Zhao, J. P. Jin, Y. J. You, W. Y. Yn, Z. T. Zhou and J. Y. Guo. Biosynthetic peptide vaccine against foot-and-mouth disease virus Recombinant and Synthetic Vaccines[J], Springer-Verlag, Berlin, Heidelberg, New York , 1994, 30-35.
[74] B. E. Clarke. S. E. Newton, A. R. Carroll, M. J. Francis, G. Appleyard, A. D. Syred, P. E. Highfield, D. J. Rowlands and F. Brown. Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein[J].Nature. 330, 1987, 381-384.
[75] S. E. Vewton, B. E. Clarke, G. Appleyard, M. J. Francis, A. R. Carroll, D. J. Rowlands, J. SkeheJ and F. Brown. Vovel antigen presentation via vaccine. In: R. M. Chanock, R A. Lerner, F. Brown and H, Ginsberg, Editors, Modern approaches to new vaccines[J], Vaccine 87 Cold Spring Harbor Laboratory, New York, 1987.
[76] R. H. Meloen, J. I. Casal, K. Dalsgaard. Langevels. Synthetic peptide vaccines: success at last. In:G. Gregoriadis, B. Mclormack and A. C. Allison, Editors, Vaccine: new generation immunological adjunants[M], Plenum, New York, 1995, 127-133.
[77] K. PeppeJ, D. Crawford. Beutler B. Atumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity[J].J. Exp.Med. 174, 1991, 1483-1489.
[78] H. Zaghouani, R. Steinman, R.Nonacs, H. Shah, W. Gerhard, C. Bona. Presentation of a viral T cell epitope expressed in the CDR 3 region of a self immunoglobulin molecule[J].Science. 259, 1993, 224-227.
[79] D. J. Capon, S. M. Chamow, J. Mordenti, S. A. Marsters, T. Gregory, H. Mitsuya, R. A. Byrn C. Lucas, F. M. Wurm, Groopman, S. Broder and D. H. Smith. Designing CD4 immunoadhesins for AIDS therapy[J].Nature 337, 1989, 525-530.
[80] T. D. Brumeanu, W. J. Swiggard, R. M. Steinman, C. A. Bona and H. Zaghouani. Efficient loading of identical viral peptide onto class II molecules by antigenized immunoglobulin and influenza virus[J] J. Exp. Med. 178, 1993, 1795-1799.
[81] H. Zaghouani, R. Steinman, R. Nonacs, H. Shah, W. Gerhard and C. Bona. Presentation of a viral T Cell epitope expressed in the CDR3 region of a self immunoglobulin molecule[J].Science.259,1993,224-227.
[82]K.Malyorzata,S.Violetta,S.Boguslaw and P.Andrzej.Inclusion bodies from recombinant bacteria as a novel system for delivery of vaccine antigen by the oral route[J].Immunology letters.91,2004,197-204.
[83]J.S.Rathel,P.R.Wood,H.F.Seow,M.W.Lightowlers.Urea/DTT solubilization of a recombinant taenia ovis antigen,45 W,expressed as a GST fusion protein results in enhanced protective immune response to the 45 Wmoiety[J].Vaccine 15,297,469-472.
[84]Kaden V,Hubert P,Strebelow qet al.Comparison of laboratory diagnostic methods for the detection of infection with the virus of classical swine fever in the early inspection phase:an experimental study[J].Berl.Munch.Tierarztl.Wochenschr.1999,112:52-57
[85]Diaz de A,Nunez J I,Garges L,et al.An RT PCR assay for the specific detection of classical swine feve virus in clinical samples[J]Vet.Res.1998,29:431-440.
[86]谢志勤,谢芝勋,廖敏等.RT-PCR检测猪瘟病毒方法的建立与应用[J].畜牧与兽医,2002,34:11-14
[87]McGoldrick A,Lowings J P,Ibata G,et al.A novel approach to the detection of classical swine fever virus by RT-PCR with a flurogenic probe(TaqMan)[J].J Virol Methods.1998,72:125-135
[88]Stegeman J A,Bouma A,Elbers A R,et aL.The leukocyte count is a valuable parameter for detecting classical swine fever[J].Diergenesskd.2000,125:511-518.
[89]Mittelhozerl C,Moser C,Tratschin J D,et al.Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent stains[J].Vet.Microbio 1.2000,74:293-308.
[90]杨仁全 猪瘟病毒E2糖蛋白抗原结构域的原核高效表达及表达产物提取[M].南京农业大学硕士论文,2001
[2]殷震,刘景华.动物病毒学,第三版[M].北京:科学出版社,1997,652-664.
[3]F.A.Murphy,C.M.Fauquet,D.H.L.Dishop,S.S.Ghabrial,A.W.Jarvis,P.Martelli,M.A.Mayo and M.D.Summers,Virus taxonomy.Sixth report of the international committee on taxonomy of viruses[J].Arch.Virol.Supp 1.10,1995,415-427.
[4]Deng R,Brock K.5'and 3'untranslated regions of pestivirus genome:primary and secondary structure analyses[J].Nucleic Acid Res.1993,21:1949-1957.
[5]Kozak M.The scanning model for translation:an update[J],J.Cell.Biol.1989,108:229-241
[6]肖明,张楚瑜,祝志展等猪瘟病毒基因组非编码区的定性、定量与结构分析[J].科学通报,2001,46:544-550.
[7]Stark,R,Rumenapf T,Meyers G,et al.Processing of pestivirus:cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus[J].J.Virol.1993,67:7088-7099.
[8]Thiel H.J,Stark R,Weiland E,et al.Hog cholera virus:molecular composition of irions from a pestvirus[J].J.Virol.1991,65:4705-4712.
[9]ELBER K,Tautz P,Becher D,et al.Processing in the pestivirus E2-NS2region:identification of protein p7 and E2p7[J].J.Virol.,70:4131-4135.
[10]Rumenapf T,Ungher C,Strauss J.H.H,et al.Processing of the envelope glycoproteins of pestivirus[J].J.Virol.1993,67:3288-3294.
[11]Elbers K,Tautz N,Becher P,et al.Processing in the pestivirus E2-p7 region:identification of protein p7 and E2p7[J].J.Virol.1996,70:4131-4135.
[12]Wensvoort GBoonstra J,Bodzinga B G.Immunoaffinity purification and characterization of the envelope protein E1 of hog cholera virus[J].J.Gen.Viro 1.1990,71:531-540.
[13]Stark R,Rumenapf T,G Meyers,et al.Genomic localization of hog cholera virus glyvoproteins[J].Virology,1990,174:286-289.
[14]Wiskerchen M M,Collett M S.Pestivirus gene expression:p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein[J].Virology.1997,165:191-199
[15]Hust M M,Himes G,Newbigin E,et al.Glycoprotein E2 of the classical swine fever virus:expression in insect cells and identification as aribonuclease[J].Virology,1994,200:588-565.
[16]Wendisch J N,S chncider R,Stark R.et al.RNase of classical swine fever virus:biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies[J].J.Virol.1996,70:352-358.
[17]韩雪清,刘湘涛,赵启祖.猪瘟病毒遗传发生关系分析[J].中国兽医科技,1999,29:3-7.
[18]王宁,付烈振,张楚瑜.猪瘟病毒囊膜糖蛋白E0的RNA酶活性及其研究进展[J].微生物学通报,1998,25:304-300.
[19]Bruschke C H 19,Hust M M,Moormann R J,et al.Glycoprotein E~(rns) of pestivirus induces apoptosis in lymphocytes of several species[J].J.Virol.1997, 71:6692-6696.
[20]Hust M M,Pauoto F E,Hoekman A,et al.nactivation of the RNase activity of glycoprotein E~(rns) of classical swine fever virus results in a cytopathogenic virus[J].J.Virol.1998,729:151-157
[21]Wendisch J M,Schncider R,Stark R,et al.RNase of classical swine fever virus:biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies[J].J.Virol.1996,70:302-308.
[22]Van Rijn PA,van Gennip H G P,Mei jier E J,et al.Epitople mapping of envelope glycoprotein E1 of hog cholera virus strain Brescia[J].J Gen.Virol.1993,74:2053-2060.
[23]Min L,Fang L,Mallery M,et al.Deletioin of structural glycoprotein E2 of classical swine fever virus strain Alfort/187 resolve a linear epitopes of monoclonal antibodies WH303 and N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum[J].J.Virol.2000,11619-11625.
[24]Yu M,Wang L F,Shiell B T,et al.Fine mapping of a C-terminal linear epitope highly conserved among the major envelope glycoprotein E2(gp51 to gp54) of different pestivirusl[J].Virololgy.1996,222:289-292.
[25]Hust M M,Moormann R J.Inhibition of pestivirus infection in cell culture by envelope proteins E~(rns) and E2 of classical swine fever virus:E~(rns) and E2 interact with different receptors[J].J Gen.Virol.1997,78:2779-2787.
[26]Collier A J,Gallego J,Klinck R,et al.A conserved RNA structure within the HCV IRES eIF3-bingding site[J].Nat.Struct.Biol.2002,9:375-380.
[27]Lowings P,Ibata G,Needham,et al.Classical swine fever virus diversity and evolution[J].J Gen.Virol.1996,77:1131-1132.
[28]赵坛,王在时,王琴等.猪瘟病毒石门株不同代次E2基因主要抗原编码区序列差异分析[J].中国兽医杂志,2002,38:7-10.
[29]付烈振,张楚瑜等.猪瘟病毒石门株E2基因序列分析[J].中国农业科学,1999,32:74-78.
[30]卞琴,李博,王在时.猪瘟病毒强弱毒株和野毒株E2全基因序列测定及比较分析[J].微生物学报,2001,41:319-328.
[31]韩雪清,李红卫,刘湘涛等.中国猪瘟兔化弱毒株(C株)兔脾组织毒土要保护性抗原E2(gp55)基因序列分析[J].畜牧兽医学报,2001,32:52-57.
[32]李红卫,涂长春,吕宗吉等.异源猪瘟病毒C株E2基因保护性抗原编码区的序列分析与比较[J].畜牧兽医学报,1998,18:112-114.
[33]李红卫,刘湘涛,李小兵.我国猪瘟病毒兔化弱毒株囊膜糖蛋白E0基因的克隆及序列测定[J].中国病毒学,1999,14:169-173.
[34]Xu J,Mendez E,Caron P R,et al.Bovine viral diarrhea virus NS3 serine protease:polyprotein cleavage sites and molecular model of an enzyme essential for pestivus replication[J]J.Virol,1997,71:5312-5322
[35]Loving CL,Brockmeier SL,Sacco RE,et al.Differential type Ⅰ interferon activation and susceptibility of dendritic cell populations to porcine arterivirus [J].Immunology,2006,76(23):154-58.
[36]Kaden V,Hubert P,Strebelow G,et al.Comparison of laboratory diagnostic methods for the detection of infection with the virus of classical swine fever in the early inspection phase:an experimental study[J].Berl.Munch.Tierarztl.Wochenschr.1999,112:52-57.
[37]Diaz de A,Sunez J I,Ganges L,et al.An RT-PCR assay for the specific detection of classical swine fever virus in clinical samples[J].Vet.Res.1998,29:431-440.
[38]Vydelingum S,Tao T,Balazse K,et al.Comparison of a reverse transcription-polymerase chain reaction assay and virus isolation for the detection of classical swine fever virus[J].Rev.Sci.Tech.1998,17:674-681.
[39]Meng Yu et al.Fine mapping of a C-terminal linear epitope highly conserved among the major envelope glycoprotein E2(gp51 to gp54) of different pestiviruses[J].Virology,1996,222:289-292.
[41]Lin M et al.Deletions of structural glycoprotein E2 of classical swine fever virus strain Alfort/187 resolve a linear epitope of monoclonal antibody WH303 and the minimal N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum[J].J Virol 2000;74(24):11619-25.
[42]G.R.Risatti et al.Identification of a novel virulence determinant within the E2structural glycoprotein of classical swine fever virus[J].Virology,2006,355:94-101.
[43]张富强,张念祖.应用噬菌体展示技术进行猪瘟病毒结构糖蛋白E2和E~(rns)中和抗原表位鉴定和比较[M].中国人民解放军军需大学博士论文,2002
[44]肖昌,余兴龙,涂长春等.利用随机肽库鉴定猪瘟病毒E2蛋白抗原表位[J].中国兽医学报,2005,25(5):449-452
[45]Ying Hua Chen et al.Candidate peptide vaccine induced protection against classical swine fever virus[J].Vaccine,2002,21:167-173
[46]Ying Hua Chen et al.Candidate multi-peptide-vaccine against classical swine fever virus induced potent immunity with serological marker[J].Vaccine,2005,23:3630-3633
[47]Ying Hua Chen et al.Spying the neutralizing epitopes on E2 N-terminal by candidate epitope-vaccines against classical swine fever virus[J].Vaccine,2006,24:4029-4034
[48]Ying Hua Chen et al.Candidate peptide-vaccine induced potent protection against CSFV and identified a principal sequential neutralizing determinant on E2[J].Vaccine,2006,24:426-434
[49]Ying Hua Chen et al.Candidate peptide-vaccines induced immunity against CSFV and identified sequential neutralizing determinants in antigenic domain A of glycoprotein E2[J].Vaccine,2006,24:1906-1913
[49]Rumenupf T,Stark R,Meyers G,et al.Structual proteins of hoglera virus expressed by vaccine virus further characterization and induction of protective immunity[J].Virol,1991,65:589-597.
[50]Hahn J.Construction of recombinant swine poxviruses and expression of classical swine fever virus E2 protein[J].Virol Methods,2001,93(1-2):49-56
[51]Berns.A live attenuated pseudorabies virus expressing envelop glycoprotein El of hog cholera virus protects swine against both the pseudorabies and hog cholera [J].Virol,1991,65:2761-2765.
[52]Van Gennip H G P,Van Rijin PA,Widojoatmodjo M 1,et al.Chimeric classical swine fever viruses containing envelope protein E~(rns) or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response[J]. Vaccine, 2001, 19:447-4}9.
[53] De Smit A J,Bouma A, Van Gennip H G P, et al.Chimeric(marker)C-stain virus induce classical protection against virulent classical swine fever virus vaccinated pigs[J].Vaccine, 2001, 19:1467-1476.
[54] Hust M M,Westra D F,Wenvoort G, et al. Glycoprotein E2 of hog cholera virus expressed in insect cell protects swine fran hog cholera[J].Virol, 1993, 67:5435-5442.
[55] Moormann R J,Bouma A. Development of classical swine fever subunit marker vaccine and companion diagnostic test[J].Vet Microbiol,2000,73(2-3):209-219.
[56] Bouma A, De Smit A J, De Jong M C, et al. Determination of the onset of the herd- immunity induced by E2 subunit vaccine against classical swine fever virus[J].Vaccine, 2000, 18(14):1374-1381.
[57] De Smith A J, Van Gennip H G P,Miedema G K W, et al. Prevention of transplacental transmission of moderate-virulent classical swine fever virus (CSFV) after as single or double vaccination with a CSFV E2 subunit vaccine [J].Vet Quart, 2000,22:150-153.
[58] Deevlf J, Laevens H, Koenen K, et al. An experimental infection with classical swine fever virus in F2 subunit marker vaccinated and in non vaccinated pigs[J]. Vaccine, 2001, 19:75-482.
[59] An S H, Song ,1 K, Kim B H, et al. Molecular biological approach for the eradication of classical swine fever in Korea[A].OIE symposium on classical swine fever (hog Cholera) C;.Birminghan, UK, 1998. 919.
[60] Yu X,Tu C,Li H, et al. DNA-mediated protection against classical swine fever virus [J].Vaccine, 2001, 19:1520-1520
[61] A. D. Guise, S. M. West and J. B. Chaudhuri. Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies[M].Mol. Biotech. 6(1996), 53-64.
[62] B. E. Fischer. Renaturation of recombinant proteins produced as inclusion bodies[J] Biotech .adv. 12(1994), 89-101.
[63] Steven B f, Bergstrom C C, Loughin T A, et al. Development of a recombination nucleoprotein-based enzyme-linked immumosorbent assay for quantification of antibodie against porcine reproductive and respiratory virus. Clin Diagn Lab Immumol. 2000. 7(4): 700-702
[64]王川庆,罗俊,丁利民,等．囊素在猪体的应用效果研究[J].中国兽医杂志,2002,38(4): 12,13
[65] D. J. Rowlands, D. V. Sangar, P. Talbot, F. Brown. Furrther evidence for multiple proteins in the foot-and-mouth disease virus particle[J].J Gen. Virol 13 (1971), 3-84.
[66] M. P. Broekhui jsen, T. Blom, M. Kottenhagen, P. H. Pounel, R. H. Meloen, S. J. Barteling, B. E. Engel Valk. Synthesis of fusion protein containing antigenic determinants of foot-and-mouth disease virus[J].Vaccine. 4 (1986), 119-124.
[67] A. M. Haberman, C. Moller, D. McGreedy and W. V. Gerhard. A large degree of functional diversity exists among helper T cells specific for the same antigenic site of influenza hemagglutinin[J].J. Immunol. 145, 1990, 3087-3094.
[68] H. Zaghouani, M. Krystal, H. Kuzu, T. Moran, H. Shah, Y. Kuzu, J. Schulman and C. Bona. Cells expressing an H chain Ig gene carrying a viral T cell epitope are lysed by specific cytotoxic Tcells[J].J. Immunol. 1489, 1992, 3604-3649.
[69] A. C. Palmenberg. Sequence alignments of picornaviral capsid proteins. In: B. L. Semler and E. Ehrenfeld, Editors, Molecular aspects of picornavirus infection and detection[J].American Society for Microbiology, Washington D C, 1989, 211-241.
[70] H. Zaghouani, S. A. Anderson, K. E. Sperber, C. Daian, R. C. Kennedy, L. Mayer and C. A. Bona. Induction of antibodies to the human immunodeficiency virus type I by immunization of baboons with immunoglobulin molecules carrying the principal neutralizing determinant of the envelope protein[J]. Proc. Net. Acad. Sci, 1995, 631-635
[71] E. W. C. Chan, H. T. along, S. C. S. Cheng .An immunoglobulin G based chimeric protein induced foot-and-mouth desease specific immune response in swine[J].Vaccine. 19 , 2000, 038-046.
[72] M. D. Winther, G. Allen, R. H. Bomford and F. Brown. Bacterially expressed antigenic peptide from foot-and-mouth disease virus capsid elicits variable immunologic responses in animals[J].J Immunol. 156, 1986, 1835-1840.
[73] Z. X. Zheng, Q. X. Xu, C. Q. Mao, H. X. Zhao, J. P. Jin, Y. J. You, W. Y. Yn, Z. T. Zhou and J. Y. Guo. Biosynthetic peptide vaccine against foot-and-mouth disease virus Recombinant and Synthetic Vaccines[J], Springer-Verlag, Berlin, Heidelberg, New York , 1994, 30-35.
[74] B. E. Clarke. S. E. Newton, A. R. Carroll, M. J. Francis, G. Appleyard, A. D. Syred, P. E. Highfield, D. J. Rowlands and F. Brown. Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein[J].Nature. 330, 1987, 381-384.
[75] S. E. Vewton, B. E. Clarke, G. Appleyard, M. J. Francis, A. R. Carroll, D. J. Rowlands, J. SkeheJ and F. Brown. Vovel antigen presentation via vaccine. In: R. M. Chanock, R A. Lerner, F. Brown and H, Ginsberg, Editors, Modern approaches to new vaccines[J], Vaccine 87 Cold Spring Harbor Laboratory, New York, 1987.
[76] R. H. Meloen, J. I. Casal, K. Dalsgaard. Langevels. Synthetic peptide vaccines: success at last. In:G. Gregoriadis, B. Mclormack and A. C. Allison, Editors, Vaccine: new generation immunological adjunants[M], Plenum, New York, 1995, 127-133.
[77] K. PeppeJ, D. Crawford. Beutler B. Atumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity[J].J. Exp.Med. 174, 1991, 1483-1489.
[78] H. Zaghouani, R. Steinman, R.Nonacs, H. Shah, W. Gerhard, C. Bona. Presentation of a viral T cell epitope expressed in the CDR 3 region of a self immunoglobulin molecule[J].Science. 259, 1993, 224-227.
[79] D. J. Capon, S. M. Chamow, J. Mordenti, S. A. Marsters, T. Gregory, H. Mitsuya, R. A. Byrn C. Lucas, F. M. Wurm, Groopman, S. Broder and D. H. Smith. Designing CD4 immunoadhesins for AIDS therapy[J].Nature 337, 1989, 525-530.
[80] T. D. Brumeanu, W. J. Swiggard, R. M. Steinman, C. A. Bona and H. Zaghouani. Efficient loading of identical viral peptide onto class II molecules by antigenized immunoglobulin and influenza virus[J] J. Exp. Med. 178, 1993, 1795-1799.
[81] H. Zaghouani, R. Steinman, R. Nonacs, H. Shah, W. Gerhard and C. Bona. Presentation of a viral T Cell epitope expressed in the CDR3 region of a self immunoglobulin molecule[J].Science.259,1993,224-227.
[82]K.Malyorzata,S.Violetta,S.Boguslaw and P.Andrzej.Inclusion bodies from recombinant bacteria as a novel system for delivery of vaccine antigen by the oral route[J].Immunology letters.91,2004,197-204.
[83]J.S.Rathel,P.R.Wood,H.F.Seow,M.W.Lightowlers.Urea/DTT solubilization of a recombinant taenia ovis antigen,45 W,expressed as a GST fusion protein results in enhanced protective immune response to the 45 Wmoiety[J].Vaccine 15,297,469-472.
[84]Kaden V,Hubert P,Strebelow qet al.Comparison of laboratory diagnostic methods for the detection of infection with the virus of classical swine fever in the early inspection phase:an experimental study[J].Berl.Munch.Tierarztl.Wochenschr.1999,112:52-57
[85]Diaz de A,Nunez J I,Garges L,et al.An RT PCR assay for the specific detection of classical swine feve virus in clinical samples[J]Vet.Res.1998,29:431-440.
[86]谢志勤,谢芝勋,廖敏等.RT-PCR检测猪瘟病毒方法的建立与应用[J].畜牧与兽医,2002,34:11-14
[87]McGoldrick A,Lowings J P,Ibata G,et al.A novel approach to the detection of classical swine fever virus by RT-PCR with a flurogenic probe(TaqMan)[J].J Virol Methods.1998,72:125-135
[88]Stegeman J A,Bouma A,Elbers A R,et aL.The leukocyte count is a valuable parameter for detecting classical swine fever[J].Diergenesskd.2000,125:511-518.
[89]Mittelhozerl C,Moser C,Tratschin J D,et al.Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent stains[J].Vet.Microbio 1.2000,74:293-308.
[90]杨仁全 猪瘟病毒E2糖蛋白抗原结构域的原核高效表达及表达产物提取[M].南京农业大学硕士论文,2001