猪瘟病毒E2基因的克隆及其在昆虫细胞中的表达
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摘要
本文利用反转录PCR（RT-PCR）和套式PCR（nPCR）技术，对流行于国内部分地区的9株猪瘟病毒（CSFV）E2基因进行克隆，并对其核苷酸及氨基酸序列的同源性进行比较及分析，绘制了系统关系发生树，以期了解近期猪瘟流行野毒主要保护性抗原E2基因的变异情况，为猪瘟的防制提供分子流行病学方面的资料。同时以1株（陕西周至株）为材料，在昆虫细胞中进行表达，为新型猪瘟基因工程亚单位疫苗的研制奠定了基础。
（1）采用RT-PCR和nPCR扩增出9株国内近期猪瘟流行野毒株的E2基因，将其分别克隆于pMD18-T载体并进行核苷酸序列测定，根据C-株，Brescia株，Alfort株及Shime株确定起始氨基酸三联体的正确位置后进行氨基酸序列推导，并进行了同源性比较，绘制了系统关系发生树。结果表明,所测9株CSFV E2基因的长度均为1324 bp，编码的氨基酸序列均包括完整信号肽序列和跨膜序列，共由442个氨基酸残基组成。可将13株(包括4株参考毒株)CSFV 分为两个组群，GXGL ，GXNN，LNAS，SDJN 4株毒株与C 株，Shimen株， Alfort株和 Brescia株同为一个群组，其E2基因间的核苷酸序列同源性为94.0%～99.4%，所推导氨基酸序列同源性为93.7%～99.5%。HNLS ，SXZZ ，HNCA ，HNXY和 HNDX 5株同为另一群组，之间核苷酸序列同源性为81.9%～99.7%，所推导氨基酸序列同源性为88.2%～99.1%。13株E2基因间的核苷酸序列同源性为81.9%～99.7%，所推导氨基酸序列同源性为88.2%～99.5%，其中 9株流行野毒与4株参考毒株之间核苷酸序列同源性为81．9%～98.9%，所推导氨基酸序列同源性为88.2%～99.5%。说明近期流行毒株的变异呈现一定的多样性。通过对主要抗原区域氨基酸位点变异进行分析，发现各毒株间抗原决定簇未发生明显的变异。
（2）对已知的E2基因进行分析后，用聚合酶链式反应（PCR）从陕西周至株克隆化E2全基因中扩增出除去3'端跨膜区的特异性片段（1 033 bp），并在其两端引入BamH I和Hind III酶切位点，将其亚克隆到杆状病毒转移载体pFastBacHTb，获得重组质粒 pFBHT-E2，转化进含穿梭载体Bacmid 的感受态细胞DH10Bac，得到含E2基因的重组载体质粒rBacmid-E2，以脂质体介导的方法将此重组载体质粒转染 sf9昆虫细胞，经SDS-PAGE和Western-blotting及ELASA等方法检测，结果表明，此E2基因在昆虫细胞中正确表达，表达的蛋白能与猪瘟阳性血清发生特异性反应。
The paper compared the differation of E2 gene nucleotide of four reference strains and 9 prevalent strains classical swine fever virus (CSFV) ; the other is E2 gene of CSFV was expressed in insect cell．
(1) The E2 gene of nine prevalent virulent strains of CSFV were amplified by reverse transcription polymerase chain reaction (RT-PCR) and the nested PCR(nPCR),Then them were cloned and sequenced, comparered with the published sequences of CSFV C-strain, Brescia，Alfort and Shime strain. The phylogenetic tree was constructed based on sequence comparion and analysis. The result indecated that there were at least two subgroups, GXNN, GXGL, LNAS , SDJN and C-strain, Brescia, Alfort, Shime strain all blonged to one subgrop. The nucleotide homology among them were 94.0% to 99.4%; and that of amino acid were 93.7% to 99.5%. HNLS, SXZZ, HNCA, HNXY and HNDX belonged to another subgroup. The nucleotide homology among them were 81.9% to 99.7％;and that of amino acid were 88.2% to 99.1%. The nucleotide homology of all the 13 strains were 81.9% to 99.7% also; and that of amino acid were 88.2% to 99.5%. It was showed epitopes of all strains were not variable obviously by analysising the variation of some main amino acid residues substitutions of E2 gene main antigenic domains．
(2) Based on E2 gene sequence date, a fragment 1033bp in E2 gene (Guangxi Yuling Strain) cloned in plasmid pMD18-T was amplified by polymerase chain reaction(PCR).The E2 fragment was subcloned into baculovirus transfer vector and recombinant baculovirus vector pFBHT-E2 was extracted.Then it was transferred into E.coli DH10Bac and recombinant bacmid was constructed. The recombinant baculovirus was obtained after the tranferation of the recombinant bacmid into sf9 cell,Thetechniques of SDS-PAGE, Western-blotting and ELISA were used to detect and identify the products expressed in sf9 cell by recombinant baculovirus. The results indicated the E2 gene was properly expressed in sf9 cell, and the expressed protein could be recognized by the postive serum of CSFV.
（1）采用RT-PCR和nPCR扩增出9株国内近期猪瘟流行野毒株的E2基因，将其分别克隆于pMD18-T载体并进行核苷酸序列测定，根据C-株，Brescia株，Alfort株及Shime株确定起始氨基酸三联体的正确位置后进行氨基酸序列推导，并进行了同源性比较，绘制了系统关系发生树。结果表明,所测9株CSFV E2基因的长度均为1324 bp，编码的氨基酸序列均包括完整信号肽序列和跨膜序列，共由442个氨基酸残基组成。可将13株(包括4株参考毒株)CSFV 分为两个组群，GXGL ，GXNN，LNAS，SDJN 4株毒株与C 株，Shimen株， Alfort株和 Brescia株同为一个群组，其E2基因间的核苷酸序列同源性为94.0%～99.4%，所推导氨基酸序列同源性为93.7%～99.5%。HNLS ，SXZZ ，HNCA ，HNXY和 HNDX 5株同为另一群组，之间核苷酸序列同源性为81.9%～99.7%，所推导氨基酸序列同源性为88.2%～99.1%。13株E2基因间的核苷酸序列同源性为81.9%～99.7%，所推导氨基酸序列同源性为88.2%～99.5%，其中 9株流行野毒与4株参考毒株之间核苷酸序列同源性为81．9%～98.9%，所推导氨基酸序列同源性为88.2%～99.5%。说明近期流行毒株的变异呈现一定的多样性。通过对主要抗原区域氨基酸位点变异进行分析，发现各毒株间抗原决定簇未发生明显的变异。
（2）对已知的E2基因进行分析后，用聚合酶链式反应（PCR）从陕西周至株克隆化E2全基因中扩增出除去3'端跨膜区的特异性片段（1 033 bp），并在其两端引入BamH I和Hind III酶切位点，将其亚克隆到杆状病毒转移载体pFastBacHTb，获得重组质粒 pFBHT-E2，转化进含穿梭载体Bacmid 的感受态细胞DH10Bac，得到含E2基因的重组载体质粒rBacmid-E2，以脂质体介导的方法将此重组载体质粒转染 sf9昆虫细胞，经SDS-PAGE和Western-blotting及ELASA等方法检测，结果表明，此E2基因在昆虫细胞中正确表达，表达的蛋白能与猪瘟阳性血清发生特异性反应。
The paper compared the differation of E2 gene nucleotide of four reference strains and 9 prevalent strains classical swine fever virus (CSFV) ; the other is E2 gene of CSFV was expressed in insect cell．
(1) The E2 gene of nine prevalent virulent strains of CSFV were amplified by reverse transcription polymerase chain reaction (RT-PCR) and the nested PCR(nPCR),Then them were cloned and sequenced, comparered with the published sequences of CSFV C-strain, Brescia，Alfort and Shime strain. The phylogenetic tree was constructed based on sequence comparion and analysis. The result indecated that there were at least two subgroups, GXNN, GXGL, LNAS , SDJN and C-strain, Brescia, Alfort, Shime strain all blonged to one subgrop. The nucleotide homology among them were 94.0% to 99.4%; and that of amino acid were 93.7% to 99.5%. HNLS, SXZZ, HNCA, HNXY and HNDX belonged to another subgroup. The nucleotide homology among them were 81.9% to 99.7％;and that of amino acid were 88.2% to 99.1%. The nucleotide homology of all the 13 strains were 81.9% to 99.7% also; and that of amino acid were 88.2% to 99.5%. It was showed epitopes of all strains were not variable obviously by analysising the variation of some main amino acid residues substitutions of E2 gene main antigenic domains．
(2) Based on E2 gene sequence date, a fragment 1033bp in E2 gene (Guangxi Yuling Strain) cloned in plasmid pMD18-T was amplified by polymerase chain reaction(PCR).The E2 fragment was subcloned into baculovirus transfer vector and recombinant baculovirus vector pFBHT-E2 was extracted.Then it was transferred into E.coli DH10Bac and recombinant bacmid was constructed. The recombinant baculovirus was obtained after the tranferation of the recombinant bacmid into sf9 cell,Thetechniques of SDS-PAGE, Western-blotting and ELISA were used to detect and identify the products expressed in sf9 cell by recombinant baculovirus. The results indicated the E2 gene was properly expressed in sf9 cell, and the expressed protein could be recognized by the postive serum of CSFV.
引文
[1] 国瑞译，陈文樾校．瘟病毒分类[J].国外兽医学—畜禽传染病,1993，13(1)：1-2．
[2] 杜念兴．猪瘟的回顾与展望[J]．中国畜禽传染病，1998，20(5)：317-319．
[3] 高彦生，马珊珊． 2002年3月国际动物疫情[J]．中国畜牧兽医， 2002，29(3)：48-50．
[4] 高彦生，邵洁环． 2002年4月国际动物疫情[J]．国外畜牧科技，2002，29(3)：51-53．
[5] 高彦生，兰乃洪．2002年6月国际动物疫情[J]．中国畜牧兽医，2002，29(4)：54-56．
[6] 李成．应用电镜技术对猪瘟病毒的研究[J]．中国兽医科技，1989，7：24-25．
[7] 王镇，闵光伟，李明义，等．猪瘟病毒形态结构与形态发生[J]．微生物学报．2000，4，（3）：237-242．
[8] 李维东译．关于猪瘟的最新知识[J]．国外兽医学—畜禽传染病，1993，13(1)：1-2．
[9] 殷震，刘景华．动物病毒学（第二版）[M]．北京：科学技术出版社，1997，54-60．
[10] 王镇，闵光伟，李明义，等．猪瘟病毒形态结构与形态发生[J]．微生物学报．2001，8（3）：227-232．
[11] 韩雪清，李红卫，刘湘涛，等．中国猪瘟兔化弱毒(C-株)兔脾组织毒部分基因的序列分析—gp55、P54、P80等基因测序方法的研究和P54基因的序列分析[J]．中国兽医科技，1998，28(6)：13-17．
[12] 韩雪清，李红卫，刘湘涛，等．中国猪瘟兔化弱毒株(C-株)兔脾组织毒主要保护性抗原E2(gp55)基因序列分析[J]．畜牧兽医学报，2001，32(1)：52-57．
[13] 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]．微生物通报，1998，25(6)：354-355．
[14] 刘湘涛，韩雪清，刘伯华，等．猪瘟流行毒Erns基因的序列分析[J]．中国农业科学，2000，33(4)：75-81．
[15] 沈同. 高级生物化学[M]．北京：高等教育出版社，1992．
[16] 周鹏程，陈建国，丁明孝，等．猪瘟病毒NS3丝氨酸蛋白酶功能区基因的克隆及其在大肠杆菌中的高效表达[J]．畜牧兽医学报，2001，32(4)：330-337．
[17] 张耀洲，张颖，吴祥莆，等．家蚕核型多角体病毒p10基因的克隆及核苷酸序列分析[J]．病毒学报，1992，8（3）：280-282．
[18] 王洵章，谢伟东，龙肇新，等．杆状病毒双基因表达转移质粒的构建[J]．病毒学报，1992，8（3）：283-285．
[19] 王健伟，姜惠英，屈建国，等．在昆虫细胞中同时表达蓝舌病毒VP3与VP7蛋白可装配成核心样颗粒[J]. 病毒学报，2000，16（2）：131-135．
[20] 健伟，姜惠英，屈建国，等．同时表达蓝舌病毒四个主要结构蛋白可装配成病毒样颗粒[J] .病毒学报，2000，16（2）：136-140．
[21] 吕鸿声．昆虫病毒分子生物学[M] ．北京：中国农业科技出版社，1998，520-529，547-580．
[22] 苗季，谭文杰，丛旭，等．中国株丙型肝炎病毒结构区蛋白在昆虫细胞中的表达及加工
[J]．病毒学报，1998，14（4）：289-295．
[23] 周玲，刘海鹰，柯越海，等．重组含有EB病毒LMP1基因的杆状病毒在昆虫细胞中的表达[J]．病毒学报，1998，14（3）：210-214．
[24] 赵炳文，李蕾，李福胜，等．人粒细胞集落刺激因子（G-CSF）受体膜外区在昆虫细胞中的表达[J]．生物工程学报，1999，15（4）：466-469．
[25] 魏婉丽，张翰，秦浚川，等．家蚕系统表达的重组人丁酰胆碱酯的生化性质[J]．中国生物化学与分子生物学报，1999，15（5）：797-801．
[26] 张传溪，姜育蕾，胡萃，等．人促红细胞生成素基因在家蚕中的高效表达[J]．生物工程学报，2000，16（1）：46-50．
[27] 于威，金勇丰，吴玉澄，等．人血小板因子IV在家蚕杆状病毒表达系统中的表达[J]．蚕业科学，2001，27（2）：100-103．
[28] 张传溪，胡萃．昆虫资源利用及其产业化的回顾与展望[J]．昆虫知识，2000，37（2）：89-95．
[29] 木村滋．昆虫生物工厂（M）．日本：工业调查会，2000，124-127。
[30] 苏慧慈，刘彦仿．原位PCR．北京：科学出版社，1997．
[31]王琴，李博，王在时，等．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(3)：320-328．
[32] 刘湘涛，赵启祖，李忠润等．猪瘟病毒和猪瘟的防制[A] ．谢庆阁，翟中和．畜禽重大疫苗研究进展[C]．北京：中国农业科技出版社，1996．
[33] 吕宗吉，涂长春，余兴龙．猪瘟病毒基因组结构与功能的研究进展[J]．中国兽医科技，2001，26(4)：8-12．
[34] 余兴龙，涂长春，李红卫，等．猪瘟病毒E2基因真核表达质粒的构建及基因疫苗的研究[J]．中国病毒学报，2000，15（3）：264-271．
[35] 崔治中．用杆状病毒为载体在昆虫细胞中表达马立克病病毒pp38基因[J]．中国病毒学报，1992，7（1）：106 -112．
[36] 韩雪清，刘湘涛，张涌，等．猪瘟病毒E2基因在Pichia pastoris中的表达及其免疫活性的初步研究[J]．生物工程学报，2002，18(2)：208 -221．
[37] 秦爱建．禽白血病病毒J亚群囊膜糖蛋白基因的生物学和生物化学特性．博士学位论文，1999，扬州大学。
[38] Edwards S, Fukusho A, Lefvre P, et al. Classical swine fever: the global sitution[J]. Vet Microbiol, 2000,73:103-119.
[39] Stegeman J A, Bouma A, Elbers AR, et al. The leukocyte count is a valuable parameter for detecting classical swine fever[J]. Tijdschr Diergeneeskd, 2000,125(17): 511-518.
[40] Changchun Tu, Zongji Lv, Hongwei Li, et al. Phylogenetic comparison of classical swine fever virus in China, Virus Res, 2001.
[41] Canal C W, Hotzel I, Almeida LL, et al. Differentiation of classical swine fever virus from ruminantpesti viruses by?reverse transcription and polymerase chain reaction (RT-PCR) [J].
Vet Microbiol, 1996,48(3-4): 373-379.
[42] Zaberezhny AD, Grebennikova TV, Kurinnov VV, et al. Differentiation between vaccine strain and field isolates of classical swine fever virus using polymerase chain reaction and restriction test[J]. Dtsch Tierarztl Wochenschr,1999,106(9): 394-397.
[43] McGoldrick A, Bensaude E, Ibata G, et al. Closed one-tube reverse transcription nested polymerase chain reaction for the detection of pestiviral RNA with fluorescent probes[J]. J Virol Methods,1999,79(1): 85-95.
[44] Mc Goldrick A, Lowings JP, Ibata G, et al. A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe (TaqMan) [J]. J Virol Methods,1998,72(2): 125-135.
[45] Barlic-Maganja D, Grom J. Highly sensitive one-tube RT-PCR andmicroplate hybridisation assay for the detection and for the discrimination of classical swine fever virus from other?pestiviruses[J]. J Virol Methods, 2001,95(1-2): 101-110.
[46] Harkness J W. Classical swine fever and its diagnosis: A current view[J]. Vet Rec, 1985, 16: 288-293.
[47] Moennig V. Characteristics of the virus. In Liess, B (Ed.) Classical swine fever and related infections. Martinus Nijhoff, Boston, Dordrecht, Lancaster, pp, 1988, 55-80.
[48] Liess B, Schurian E. Inaktivierende wirkung von TEGO 51/15 DL im suspension svers -uchmit zwei virusarten der Toga-Gruppe: de Euro paischen schweinepest and der Bovinen virusdiarr hoe-Mucosal Disease[J]. Archir Fur lebensmittelhygiene, 1973, 24: 154-157.
[49] Russell A D, Hugo W B. Chemical disinfectants. In: Linton, AH,Hugo WB, Rus sell AD(Eds), Disinfection in veterinary and farm animal practice. Blackwell scientific publications, Oxford, pp, 1987,12-42.
[50] Freitas T R, Caldas L A, Rebello M A. Protaglandin A1 inhibits replication of classical swine fever virus[J]. Mem Inst Oswaldo Cruz, 1998, 93(6):815-818.
[51] Moenning V. The hog cholera virus[J]. Comp Immun Microbiol Infect Disease, 1992, 15(3): 189-201.
[52] TrautweinG. Pathology and pathogenesis of the diease, Martins Nijhoff publishing. Boston, USA, 1988, 27-54.
[53] Flores E F, et al. Swine and ruminant pestiviruses require the same cellular factors to enter bovine cells[J]. J Gen Virol, 1996, 77:1295-1303.
[54] Hulst M M , Moormann R J M. Inhibition of pestivirus infection in cell culture by envelope protein Erns and E2 of classical swine fever virus: Erns and E2 interact with different receptor[J]s. J Gen Virol, 1997, 78:2779-2787.
[55] Hulst M M, Van Gennip H G P,Moormann R J M. Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein Erns[J]. J Virol, 2000, 74(20): 9553-9561.
[56] Pestova T V, Shatsky I N, Fletcher S P, et al. A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of
hepatitis C and classical swine fever virus RNAs[J]. Genes Dev, 1998, 12 (1):67-83.
[57] Rumenapf T, Ungher G, Strauss J H, H J Thiel. Processing of the envelope glycoprote -ins of pestivirus[J]. J Virol, 1993, 67:3288-3294.
[58] Moennig V. Introduction to classical swine fever: virus, disease and control policy[J]. Vet Microbiol. 2000,73(2-3): 93-102.
[59] Moenning V , P G W Plagemann. The pestivirus. Adv Virus Res, 1992, 41:53-98.
[60] Luckow V A, Lee S C, Barry G F, et al. Efficient generation infectious recombinant baculovirus by site-specific transposon-mediated insertion of foreign gene into baculovirus genome propagated in Escherichia coli[J]. J Virol,1993, 67:4566-4579.
[61] Moenning V. The hog cholera virus[J]. Comp Immun Microbiol Infect Disease,1992, 15(3): 189-201.
[62] Konig M, Lengs feld T, T Pauly, R Stark and H J Thiel. Classical swine fever virus: ind -ependent induction of protective immunity by two structural glycoprotein[J]. J. Virol, 1995, 69:6479-6486.
[63] Wensvoort G. Topographical and functional mapping of epitopes on hog cholera virus with monoclonal antibodies[J]. J Gen Virol, 1989,70:2865-2876.
[64] Van Rijn PA, van Gennip HGP, de Meijer E J, et al. Epitope mapping of envelope glycoprotein E1 of hog cholera virus strain Brecia[J]. J Gen Virol, 1993,74:2053-2060.
[65] Van Rijn PA, vans Gennip HG, Moormann RJ. An experimental marker vaccine and accompanying serological diagnostic test both based on envelope glycoprotein E2 of classical swine fever virus (CSFV) [J]. Vaccine, 1999,17(5): 433-440.
[66] Van Rijn P A, Miedema GKW, Wensvoort G, et al. Antigenic structure of envelope enviloprotein E1 of hog cholera virus[J]. Journal of virology,1994, 68(6):3934-3942.
[67] Schneider R, Unger G, Stark R ,et al. Identification of a structural glycoprotein of an RNA virus as a ribonuclease[J]. Science, 1993, 261: 1169-1171.
[68] Bruschke C J M, Hulst M M, Moormann R J M, et al. Glycoprotein Erns of pestivirus induces apoptosis in lymphocytes of several species[J]. J Virol, 1997,71:6692-6696.
[69] Meyers G, Saalmuller A, Buttner M. Mutations abrogating the RNase activity in glycoprotein Erns of the pestivirus classical swine fever virus[J]. J Virol, 1999,73(12): 10224-10235.
[70] Stark R. Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus[J]. J Virol.1993, 67:7088-7095.
[71] Rumenapf T. structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity[J]. J Virol, 1991,67:589-597.
[72] Tratschin JD, Moser C, Ruggli N, et al. Classical swine fever virus leader proteinase Npro is not required for viral replication in cell culture[J]. J Virol, 1998,72(9): 7681-7684.
[73] Rumenapf T, Stark R, Heimann M, et al. N-terminal proteinase of pestivirus: identification of putative catalytic residues by site-directed mutagenesis[J]. J Virol, 1998,72(3): 2544-2547.
[74] Behrens S E, Grassmann C W, Thiel H J， et al. Characterization of an autonomous
subgenomic pestivirus RNA replicon[J]. J Virol, 1998,72:2364-2372.
[75] Bakkali Kassimi L, Gonzague M, Boutrouille A, et al. Expression and characterization of part of hog cholera virus non-structural proteins[J]. Zentralbl Veterinarmed , 1996,43(3): 167-177.
[76] Warrener P, Collectt MS. Pestivirus NS3 (p80) protein possesses RNA helicase activity[J]. J Virol, 1995,69(3): 1720-1726.
[77] Paton D J, McGoldrick A, Greiser-Wilke I, et al. Genetic typing of classical swine fever virus[J]. Vet Microbiol, 2000,73(2-3): 135-157.
[78] Steffeus S, Thiel H-J, Behrens S-E, et al. The RNA-dependent RNA polymerase of different members of the family flaviviridae exhibit similar properities in vitro[J]. J Gen Virol,1999, 80: 2583-2590.
[79] Lowing P, Ibrate G, Needham J, et al. Classical swine fever virus diversity and evolution[J]. J Gen Virology, 1996,77:1311-1321.
[80] Rumenapf T, Stark, R . G Meyer and H J Thiel. Structural proteins of hog cholera virus expressed by vaccinia virus: Further characterization and induction of protecting immunity[J]. J Virol, 1991, 67:589-597.
[81] Van Rijn P A, G Wensvoort, E de Kluyver, M M Hust, et al. Live attenuated pseudorabies virus expressing envelop glycoprotein E1 of Hog cholera virus protects swine against pseudiverirys and hog cholera virus[J]. J Virol, 1991, 65:2761-2765.
[82] Berns A, Moonrman R.Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protect swine against both pseudorabies and hog cholera[J]. J virol,1991,65:2761-2765.
[83] Peeters B, Bienkowska-Szewczyk K, Hulst M, et al. Biologically safe, non-transmissible pseudorabies virus vector vaccine protects?pigs against both Aujeszky’s disease and classical swine fever[J]. J Gen Virol,1997,78(Pt 12):3311-3315.
[84] Mulder W A M, priem J, GlazenburaK L, et al. Virulence and pathogenesis of non virulent and virulent strains of pseudorabies virus expressing envelope glyoprotein E1 of hog cholera virus[J].J Gen virol,1994,75:117-124.
[85] Hulst M M, et al. Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera[J]. J Virol, 1993, 67: 5435-5442.
[86] Bouma A, De Smit A J, De Jong M C M, et al. Determination of the onset of the herd- immunity induced by the E2 sub-unit vaccine against classical swine fever virus[J]. Vaccine, 2000, 18:1374-1381.
[87] Bouma A, de Smith A J, De Klui jver E P, et al. Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus[J]. Vet Microbiol,1999, 66(2):101-104.
[88] De Smit A J, Bouma A, Van Gennip H G P, et al. Chimeric (marker)C-strain viruses induce clinical protection against virulent classical swine fever virus (CSFV) and reduce transmission of CSFV between vaccinated pigs[J]. Vaccine, 201, 19:1467-1476.
[89] Moormann R J M, Bouma A, Kramp S J A, et al. Development of a classical swine fever subunit marker vaccine and companion diagnostic test[J]. J Virol, 2001, 132:763-770.
[90] Widjojoat Moajo M N, Van Gennip H G P, Bouma A. Classical swine fever virus Erns deletion mutants: transcomplementation and potential use as nontransmissible, modifie -d, live-attenuated marker vaccines. J Virology, 2000, 74(7): 2973-2980.
[91]Van Gennip HG, van Rijn PA, Widjojoatmodjo MN 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. 2000,19(4-5): 447-459.
[92] Dewulf,Theis S, Daniel H. Expression and functional charaterization of the mammali an intesinal peptide transporter PepT1 in the methylotropic yeast[J]. Microbiology, 2001, 80:367-388.
[93] Yu X, Tu C, Li H, et al. DNA-mediated protection against classical swine fever virus [J], Vaccine, 2001, 19:1520-1535.
[94] Smith G E, Vlak J M, Summers M D. Physical analysis of AcMNPV transcripts for polyhedrin and 10,000 molecular-weight protein[J]. Journal of Virology, 1983, 45: 215-225.
[95] Fra K I.Classification and Namenclature of Viruses, Sixth repont of the Znte mational Committee on Tazonormy of Viruses[M]. Wien and New york: Springer verlag.1997.
[96] Luckow V A, Summers M D. Trends in the development of Baculovirus expression vectors[J]. Biotechnology,1988,6: 47.
[97] Ayres M D, Howard S C, Kuzio J, et al. The complete DNA sequence of Autographa californica nuclear polyhedrosis virus[J]. Virology,1994, 202: 586-605.
[98] Kuzio J, Rohel O Z, Curry C J, et al. Nucleotide sequence of the p10 polypeptite gene of Autographa californica nuclear polyhedrosis in insect cells with a baculovirus[J]. Virology, 1984,139: 307-314.
[99] Roelvink P W, Van Meer M M, de Kort C A, et al. Dissimilar expression of Autographa californica multiple nucleaocapsid nuclear polyhedrosis virus polyhedrin and p10 genes[J]. Joural General Virology, 1992,73(6):1481-1489.
[100] Miller L K. Baculoviruses as gene expression vectors [J]. Annual Review of Microbiology, 1988, 42: 177-199.
[101] Vlak J M, Kilnenberg F A, Zaal K J, et al. Functional studies on the p10 gene of californica nuclear polyhedrosis virus using a recominant expressing a p10-bata-galactosidase fusion gene[J]. Journal of General Virology, 1988,69; 765-776.
[102] Weyer U, Knight S, Possee R D. Analysis of very late gene expression by californica nuclear polyhedrosis virus and the further development of nultiple expression vectors[J]. Journal of General Virology, 1990. 71: 1525-1534.
[103] Smith G E, Ju G, Ericson B L, Moschera J, et al. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector[J]. Procdkings of National Academy Sciences USA,1985,82:8404-8408.
[104] Luckow V A, Summers M D. High-lever expression of nonfused foreign genes with Autographa californica nuclearvirus expression vectors[J]. Virology, 1989,170: 31-39.
[105] Matsuura Y, Possee R D,Bishop D H. Baculovirus expression vectors: The requirement for high level expression of proteins, including glycoprotein[J]. Journal of General Virology, 1987,68:1232-1250.
[106] Reis U, Blum B, Von Specht B U, Domdey H, Collins J, Antibody production in silk worm cells and silkworm larvae infected with a dual recombination Bombwz orinuclea polyhedrosis virus[J]. Biotechnology, 1992,10:910-912.
[107] Belyaev A S, Roy P. Development of baculovirus tripe and quadruple expression vector: co-expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells[J].Nucleic acids Research,1993,21(5): 1219-1223.
[108] Ayres M D, Howard S C, Kuzio J, et al. The complete DNA sequence of Autographa californica nuclear polyhedrosis virus[J]. Virology,1994, 202:586-605.
[109] Chajanovsky N. Functional expression of an alpha anti-insect scorption neuroorin in insect cells and lepidopterons larvae[J]. FEBS letters, 1995,376:181-184.
[110] Summer M D, Smith G E. A manual of methods for baculovirus vectors and insect cell culture priducedures. Texas Agricultural Experiment station Bulletin.1987,1555.
[111] Maeda S, Kawai T, Obiata M, et al. Production of human alpha-interferon in silkworm using a baculovirus vector[J]. Nature,1985,315:592-594.
[112] Matsuura Y, Possee R D, Bishop D H. Expression of the S-coded genes of lymphocytic choriomeningitis arenavirus using a baclovirus vector[J]. Journal of General Virology,1986, 67: 1515-1529.
[113] Matsuura Y, Possee R D. Bishop D H. Baculovirus expression vectors: The requirement for high level expression of proteins, including glycoprotein[J]. Journal of General Virology, 1987,68:1232-1250.
[114] Lanford R E, Luckow V, Kennedy R C , et al. Expression and characterization of hepatitis B virus surface antigen polypeptides in insect cells with a baculovirus expression system[J]. Journal of Virology, 1989, 63:1549-1557.
[115] Gomi S, Zhou E Z, et al. Delection analysis of eighteen late gene expression factor gene homologues of baculovirus BnNPV[J].Virology,1997,230:35-47.
[116] Manji G A. Baclovirus inhibitor of apoptosis functions at or upstream of the apoptotic suppressor p35 to prevent programmed cell death [J]. Virol,1997,71: 4509-4516.
[117] kihiro U. Improvement of baculovirus expression system(A).prospects for the Development of Insect Factories(M).Japan: Institute of Insect and Animal Science national Institute of Agrobiological Science,2001.57-63.
[118] Muneta Y. Production and utilization of pig cytokines using baculovirus insect expression systems(A).Prospects for the Development of Insect Factories(M).Japan: Institute of Insect and Animal Sciences National Institute of Agrobiological Science,2001,25-33.
[119] Ohura M. The 5th NISES/COE International symposium: Research for the utilization of
insect properties-prospect for the development of new materials?. Tsukuba, Japan.2000.
[120] Ellis A E.Meeting the requirements for delayed release of oral vaccines for fish[J].Journal of Applied Ichthyology,1998,14:149-152.
[121] Inceoglu A B, Kamita S G, Hinton A C, et al. Recombinant baculoviruses for insect control[J].Pest Management Science,2001,57:981-987.
[122] Flores E F, et al. Swine and ruminant pestiviruses require the same cellular factors to enter bovine cells[J]. J Gen Virol, 1996, 77:1295-1303.
[123] Van Ri jn P A,Van gennip H G P, Meijer E J, et al .Epitope mapping of envelop glyprotein E1 of hog cholera virus strain Brescia[J]. Journal of general virology,1993,74:2053-2060.
[124] Kozak M. The scanning model for translation: An update[J]. Jouranal of Cell Biology,1989,108(2):229-241.
[125] Luckow,Y. A.and Summer, M D. Singnals important for high-lever expression of foreign gene in Autographa californica nuclear polyhedrosis virus expression vector[J].Virology,1988,167:56-71.
[126 ]Hammond J M, Mccoy R J, Jansen E S ,et al. Vaccination with a single dose of a recombinant porcine adenovirus expressing the classical swine fever virus gp55(E2) gene protects pigs against classical swine fever[J].Vaccine,2000,18(1-12):1040-1050.
[2] 杜念兴．猪瘟的回顾与展望[J]．中国畜禽传染病，1998，20(5)：317-319．
[3] 高彦生，马珊珊． 2002年3月国际动物疫情[J]．中国畜牧兽医， 2002，29(3)：48-50．
[4] 高彦生，邵洁环． 2002年4月国际动物疫情[J]．国外畜牧科技，2002，29(3)：51-53．
[5] 高彦生，兰乃洪．2002年6月国际动物疫情[J]．中国畜牧兽医，2002，29(4)：54-56．
[6] 李成．应用电镜技术对猪瘟病毒的研究[J]．中国兽医科技，1989，7：24-25．
[7] 王镇，闵光伟，李明义，等．猪瘟病毒形态结构与形态发生[J]．微生物学报．2000，4，（3）：237-242．
[8] 李维东译．关于猪瘟的最新知识[J]．国外兽医学—畜禽传染病，1993，13(1)：1-2．
[9] 殷震，刘景华．动物病毒学（第二版）[M]．北京：科学技术出版社，1997，54-60．
[10] 王镇，闵光伟，李明义，等．猪瘟病毒形态结构与形态发生[J]．微生物学报．2001，8（3）：227-232．
[11] 韩雪清，李红卫，刘湘涛，等．中国猪瘟兔化弱毒(C-株)兔脾组织毒部分基因的序列分析—gp55、P54、P80等基因测序方法的研究和P54基因的序列分析[J]．中国兽医科技，1998，28(6)：13-17．
[12] 韩雪清，李红卫，刘湘涛，等．中国猪瘟兔化弱毒株(C-株)兔脾组织毒主要保护性抗原E2(gp55)基因序列分析[J]．畜牧兽医学报，2001，32(1)：52-57．
[13] 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]．微生物通报，1998，25(6)：354-355．
[14] 刘湘涛，韩雪清，刘伯华，等．猪瘟流行毒Erns基因的序列分析[J]．中国农业科学，2000，33(4)：75-81．
[15] 沈同. 高级生物化学[M]．北京：高等教育出版社，1992．
[16] 周鹏程，陈建国，丁明孝，等．猪瘟病毒NS3丝氨酸蛋白酶功能区基因的克隆及其在大肠杆菌中的高效表达[J]．畜牧兽医学报，2001，32(4)：330-337．
[17] 张耀洲，张颖，吴祥莆，等．家蚕核型多角体病毒p10基因的克隆及核苷酸序列分析[J]．病毒学报，1992，8（3）：280-282．
[18] 王洵章，谢伟东，龙肇新，等．杆状病毒双基因表达转移质粒的构建[J]．病毒学报，1992，8（3）：283-285．
[19] 王健伟，姜惠英，屈建国，等．在昆虫细胞中同时表达蓝舌病毒VP3与VP7蛋白可装配成核心样颗粒[J]. 病毒学报，2000，16（2）：131-135．
[20] 健伟，姜惠英，屈建国，等．同时表达蓝舌病毒四个主要结构蛋白可装配成病毒样颗粒[J] .病毒学报，2000，16（2）：136-140．
[21] 吕鸿声．昆虫病毒分子生物学[M] ．北京：中国农业科技出版社，1998，520-529，547-580．
[22] 苗季，谭文杰，丛旭，等．中国株丙型肝炎病毒结构区蛋白在昆虫细胞中的表达及加工
[J]．病毒学报，1998，14（4）：289-295．
[23] 周玲，刘海鹰，柯越海，等．重组含有EB病毒LMP1基因的杆状病毒在昆虫细胞中的表达[J]．病毒学报，1998，14（3）：210-214．
[24] 赵炳文，李蕾，李福胜，等．人粒细胞集落刺激因子（G-CSF）受体膜外区在昆虫细胞中的表达[J]．生物工程学报，1999，15（4）：466-469．
[25] 魏婉丽，张翰，秦浚川，等．家蚕系统表达的重组人丁酰胆碱酯的生化性质[J]．中国生物化学与分子生物学报，1999，15（5）：797-801．
[26] 张传溪，姜育蕾，胡萃，等．人促红细胞生成素基因在家蚕中的高效表达[J]．生物工程学报，2000，16（1）：46-50．
[27] 于威，金勇丰，吴玉澄，等．人血小板因子IV在家蚕杆状病毒表达系统中的表达[J]．蚕业科学，2001，27（2）：100-103．
[28] 张传溪，胡萃．昆虫资源利用及其产业化的回顾与展望[J]．昆虫知识，2000，37（2）：89-95．
[29] 木村滋．昆虫生物工厂（M）．日本：工业调查会，2000，124-127。
[30] 苏慧慈，刘彦仿．原位PCR．北京：科学出版社，1997．
[31]王琴，李博，王在时，等．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(3)：320-328．
[32] 刘湘涛，赵启祖，李忠润等．猪瘟病毒和猪瘟的防制[A] ．谢庆阁，翟中和．畜禽重大疫苗研究进展[C]．北京：中国农业科技出版社，1996．
[33] 吕宗吉，涂长春，余兴龙．猪瘟病毒基因组结构与功能的研究进展[J]．中国兽医科技，2001，26(4)：8-12．
[34] 余兴龙，涂长春，李红卫，等．猪瘟病毒E2基因真核表达质粒的构建及基因疫苗的研究[J]．中国病毒学报，2000，15（3）：264-271．
[35] 崔治中．用杆状病毒为载体在昆虫细胞中表达马立克病病毒pp38基因[J]．中国病毒学报，1992，7（1）：106 -112．
[36] 韩雪清，刘湘涛，张涌，等．猪瘟病毒E2基因在Pichia pastoris中的表达及其免疫活性的初步研究[J]．生物工程学报，2002，18(2)：208 -221．
[37] 秦爱建．禽白血病病毒J亚群囊膜糖蛋白基因的生物学和生物化学特性．博士学位论文，1999，扬州大学。
[38] Edwards S, Fukusho A, Lefvre P, et al. Classical swine fever: the global sitution[J]. Vet Microbiol, 2000,73:103-119.
[39] Stegeman J A, Bouma A, Elbers AR, et al. The leukocyte count is a valuable parameter for detecting classical swine fever[J]. Tijdschr Diergeneeskd, 2000,125(17): 511-518.
[40] Changchun Tu, Zongji Lv, Hongwei Li, et al. Phylogenetic comparison of classical swine fever virus in China, Virus Res, 2001.
[41] Canal C W, Hotzel I, Almeida LL, et al. Differentiation of classical swine fever virus from ruminantpesti viruses by?reverse transcription and polymerase chain reaction (RT-PCR) [J].
Vet Microbiol, 1996,48(3-4): 373-379.
[42] Zaberezhny AD, Grebennikova TV, Kurinnov VV, et al. Differentiation between vaccine strain and field isolates of classical swine fever virus using polymerase chain reaction and restriction test[J]. Dtsch Tierarztl Wochenschr,1999,106(9): 394-397.
[43] McGoldrick A, Bensaude E, Ibata G, et al. Closed one-tube reverse transcription nested polymerase chain reaction for the detection of pestiviral RNA with fluorescent probes[J]. J Virol Methods,1999,79(1): 85-95.
[44] Mc Goldrick A, Lowings JP, Ibata G, et al. A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe (TaqMan) [J]. J Virol Methods,1998,72(2): 125-135.
[45] Barlic-Maganja D, Grom J. Highly sensitive one-tube RT-PCR andmicroplate hybridisation assay for the detection and for the discrimination of classical swine fever virus from other?pestiviruses[J]. J Virol Methods, 2001,95(1-2): 101-110.
[46] Harkness J W. Classical swine fever and its diagnosis: A current view[J]. Vet Rec, 1985, 16: 288-293.
[47] Moennig V. Characteristics of the virus. In Liess, B (Ed.) Classical swine fever and related infections. Martinus Nijhoff, Boston, Dordrecht, Lancaster, pp, 1988, 55-80.
[48] Liess B, Schurian E. Inaktivierende wirkung von TEGO 51/15 DL im suspension svers -uchmit zwei virusarten der Toga-Gruppe: de Euro paischen schweinepest and der Bovinen virusdiarr hoe-Mucosal Disease[J]. Archir Fur lebensmittelhygiene, 1973, 24: 154-157.
[49] Russell A D, Hugo W B. Chemical disinfectants. In: Linton, AH,Hugo WB, Rus sell AD(Eds), Disinfection in veterinary and farm animal practice. Blackwell scientific publications, Oxford, pp, 1987,12-42.
[50] Freitas T R, Caldas L A, Rebello M A. Protaglandin A1 inhibits replication of classical swine fever virus[J]. Mem Inst Oswaldo Cruz, 1998, 93(6):815-818.
[51] Moenning V. The hog cholera virus[J]. Comp Immun Microbiol Infect Disease, 1992, 15(3): 189-201.
[52] TrautweinG. Pathology and pathogenesis of the diease, Martins Nijhoff publishing. Boston, USA, 1988, 27-54.
[53] Flores E F, et al. Swine and ruminant pestiviruses require the same cellular factors to enter bovine cells[J]. J Gen Virol, 1996, 77:1295-1303.
[54] Hulst M M , Moormann R J M. Inhibition of pestivirus infection in cell culture by envelope protein Erns and E2 of classical swine fever virus: Erns and E2 interact with different receptor[J]s. J Gen Virol, 1997, 78:2779-2787.
[55] Hulst M M, Van Gennip H G P,Moormann R J M. Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein Erns[J]. J Virol, 2000, 74(20): 9553-9561.
[56] Pestova T V, Shatsky I N, Fletcher S P, et al. A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of
hepatitis C and classical swine fever virus RNAs[J]. Genes Dev, 1998, 12 (1):67-83.
[57] Rumenapf T, Ungher G, Strauss J H, H J Thiel. Processing of the envelope glycoprote -ins of pestivirus[J]. J Virol, 1993, 67:3288-3294.
[58] Moennig V. Introduction to classical swine fever: virus, disease and control policy[J]. Vet Microbiol. 2000,73(2-3): 93-102.
[59] Moenning V , P G W Plagemann. The pestivirus. Adv Virus Res, 1992, 41:53-98.
[60] Luckow V A, Lee S C, Barry G F, et al. Efficient generation infectious recombinant baculovirus by site-specific transposon-mediated insertion of foreign gene into baculovirus genome propagated in Escherichia coli[J]. J Virol,1993, 67:4566-4579.
[61] Moenning V. The hog cholera virus[J]. Comp Immun Microbiol Infect Disease,1992, 15(3): 189-201.
[62] Konig M, Lengs feld T, T Pauly, R Stark and H J Thiel. Classical swine fever virus: ind -ependent induction of protective immunity by two structural glycoprotein[J]. J. Virol, 1995, 69:6479-6486.
[63] Wensvoort G. Topographical and functional mapping of epitopes on hog cholera virus with monoclonal antibodies[J]. J Gen Virol, 1989,70:2865-2876.
[64] Van Rijn PA, van Gennip HGP, de Meijer E J, et al. Epitope mapping of envelope glycoprotein E1 of hog cholera virus strain Brecia[J]. J Gen Virol, 1993,74:2053-2060.
[65] Van Rijn PA, vans Gennip HG, Moormann RJ. An experimental marker vaccine and accompanying serological diagnostic test both based on envelope glycoprotein E2 of classical swine fever virus (CSFV) [J]. Vaccine, 1999,17(5): 433-440.
[66] Van Rijn P A, Miedema GKW, Wensvoort G, et al. Antigenic structure of envelope enviloprotein E1 of hog cholera virus[J]. Journal of virology,1994, 68(6):3934-3942.
[67] Schneider R, Unger G, Stark R ,et al. Identification of a structural glycoprotein of an RNA virus as a ribonuclease[J]. Science, 1993, 261: 1169-1171.
[68] Bruschke C J M, Hulst M M, Moormann R J M, et al. Glycoprotein Erns of pestivirus induces apoptosis in lymphocytes of several species[J]. J Virol, 1997,71:6692-6696.
[69] Meyers G, Saalmuller A, Buttner M. Mutations abrogating the RNase activity in glycoprotein Erns of the pestivirus classical swine fever virus[J]. J Virol, 1999,73(12): 10224-10235.
[70] Stark R. Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus[J]. J Virol.1993, 67:7088-7095.
[71] Rumenapf T. structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity[J]. J Virol, 1991,67:589-597.
[72] Tratschin JD, Moser C, Ruggli N, et al. Classical swine fever virus leader proteinase Npro is not required for viral replication in cell culture[J]. J Virol, 1998,72(9): 7681-7684.
[73] Rumenapf T, Stark R, Heimann M, et al. N-terminal proteinase of pestivirus: identification of putative catalytic residues by site-directed mutagenesis[J]. J Virol, 1998,72(3): 2544-2547.
[74] Behrens S E, Grassmann C W, Thiel H J， et al. Characterization of an autonomous
subgenomic pestivirus RNA replicon[J]. J Virol, 1998,72:2364-2372.
[75] Bakkali Kassimi L, Gonzague M, Boutrouille A, et al. Expression and characterization of part of hog cholera virus non-structural proteins[J]. Zentralbl Veterinarmed , 1996,43(3): 167-177.
[76] Warrener P, Collectt MS. Pestivirus NS3 (p80) protein possesses RNA helicase activity[J]. J Virol, 1995,69(3): 1720-1726.
[77] Paton D J, McGoldrick A, Greiser-Wilke I, et al. Genetic typing of classical swine fever virus[J]. Vet Microbiol, 2000,73(2-3): 135-157.
[78] Steffeus S, Thiel H-J, Behrens S-E, et al. The RNA-dependent RNA polymerase of different members of the family flaviviridae exhibit similar properities in vitro[J]. J Gen Virol,1999, 80: 2583-2590.
[79] Lowing P, Ibrate G, Needham J, et al. Classical swine fever virus diversity and evolution[J]. J Gen Virology, 1996,77:1311-1321.
[80] Rumenapf T, Stark, R . G Meyer and H J Thiel. Structural proteins of hog cholera virus expressed by vaccinia virus: Further characterization and induction of protecting immunity[J]. J Virol, 1991, 67:589-597.
[81] Van Rijn P A, G Wensvoort, E de Kluyver, M M Hust, et al. Live attenuated pseudorabies virus expressing envelop glycoprotein E1 of Hog cholera virus protects swine against pseudiverirys and hog cholera virus[J]. J Virol, 1991, 65:2761-2765.
[82] Berns A, Moonrman R.Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protect swine against both pseudorabies and hog cholera[J]. J virol,1991,65:2761-2765.
[83] Peeters B, Bienkowska-Szewczyk K, Hulst M, et al. Biologically safe, non-transmissible pseudorabies virus vector vaccine protects?pigs against both Aujeszky’s disease and classical swine fever[J]. J Gen Virol,1997,78(Pt 12):3311-3315.
[84] Mulder W A M, priem J, GlazenburaK L, et al. Virulence and pathogenesis of non virulent and virulent strains of pseudorabies virus expressing envelope glyoprotein E1 of hog cholera virus[J].J Gen virol,1994,75:117-124.
[85] Hulst M M, et al. Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera[J]. J Virol, 1993, 67: 5435-5442.
[86] Bouma A, De Smit A J, De Jong M C M, et al. Determination of the onset of the herd- immunity induced by the E2 sub-unit vaccine against classical swine fever virus[J]. Vaccine, 2000, 18:1374-1381.
[87] Bouma A, de Smith A J, De Klui jver E P, et al. Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus[J]. Vet Microbiol,1999, 66(2):101-104.
[88] De Smit A J, Bouma A, Van Gennip H G P, et al. Chimeric (marker)C-strain viruses induce clinical protection against virulent classical swine fever virus (CSFV) and reduce transmission of CSFV between vaccinated pigs[J]. Vaccine, 201, 19:1467-1476.
[89] Moormann R J M, Bouma A, Kramp S J A, et al. Development of a classical swine fever subunit marker vaccine and companion diagnostic test[J]. J Virol, 2001, 132:763-770.
[90] Widjojoat Moajo M N, Van Gennip H G P, Bouma A. Classical swine fever virus Erns deletion mutants: transcomplementation and potential use as nontransmissible, modifie -d, live-attenuated marker vaccines. J Virology, 2000, 74(7): 2973-2980.
[91]Van Gennip HG, van Rijn PA, Widjojoatmodjo MN 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. 2000,19(4-5): 447-459.
[92] Dewulf,Theis S, Daniel H. Expression and functional charaterization of the mammali an intesinal peptide transporter PepT1 in the methylotropic yeast[J]. Microbiology, 2001, 80:367-388.
[93] Yu X, Tu C, Li H, et al. DNA-mediated protection against classical swine fever virus [J], Vaccine, 2001, 19:1520-1535.
[94] Smith G E, Vlak J M, Summers M D. Physical analysis of AcMNPV transcripts for polyhedrin and 10,000 molecular-weight protein[J]. Journal of Virology, 1983, 45: 215-225.
[95] Fra K I.Classification and Namenclature of Viruses, Sixth repont of the Znte mational Committee on Tazonormy of Viruses[M]. Wien and New york: Springer verlag.1997.
[96] Luckow V A, Summers M D. Trends in the development of Baculovirus expression vectors[J]. Biotechnology,1988,6: 47.
[97] Ayres M D, Howard S C, Kuzio J, et al. The complete DNA sequence of Autographa californica nuclear polyhedrosis virus[J]. Virology,1994, 202: 586-605.
[98] Kuzio J, Rohel O Z, Curry C J, et al. Nucleotide sequence of the p10 polypeptite gene of Autographa californica nuclear polyhedrosis in insect cells with a baculovirus[J]. Virology, 1984,139: 307-314.
[99] Roelvink P W, Van Meer M M, de Kort C A, et al. Dissimilar expression of Autographa californica multiple nucleaocapsid nuclear polyhedrosis virus polyhedrin and p10 genes[J]. Joural General Virology, 1992,73(6):1481-1489.
[100] Miller L K. Baculoviruses as gene expression vectors [J]. Annual Review of Microbiology, 1988, 42: 177-199.
[101] Vlak J M, Kilnenberg F A, Zaal K J, et al. Functional studies on the p10 gene of californica nuclear polyhedrosis virus using a recominant expressing a p10-bata-galactosidase fusion gene[J]. Journal of General Virology, 1988,69; 765-776.
[102] Weyer U, Knight S, Possee R D. Analysis of very late gene expression by californica nuclear polyhedrosis virus and the further development of nultiple expression vectors[J]. Journal of General Virology, 1990. 71: 1525-1534.
[103] Smith G E, Ju G, Ericson B L, Moschera J, et al. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector[J]. Procdkings of National Academy Sciences USA,1985,82:8404-8408.
[104] Luckow V A, Summers M D. High-lever expression of nonfused foreign genes with Autographa californica nuclearvirus expression vectors[J]. Virology, 1989,170: 31-39.
[105] Matsuura Y, Possee R D,Bishop D H. Baculovirus expression vectors: The requirement for high level expression of proteins, including glycoprotein[J]. Journal of General Virology, 1987,68:1232-1250.
[106] Reis U, Blum B, Von Specht B U, Domdey H, Collins J, Antibody production in silk worm cells and silkworm larvae infected with a dual recombination Bombwz orinuclea polyhedrosis virus[J]. Biotechnology, 1992,10:910-912.
[107] Belyaev A S, Roy P. Development of baculovirus tripe and quadruple expression vector: co-expression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells[J].Nucleic acids Research,1993,21(5): 1219-1223.
[108] Ayres M D, Howard S C, Kuzio J, et al. The complete DNA sequence of Autographa californica nuclear polyhedrosis virus[J]. Virology,1994, 202:586-605.
[109] Chajanovsky N. Functional expression of an alpha anti-insect scorption neuroorin in insect cells and lepidopterons larvae[J]. FEBS letters, 1995,376:181-184.
[110] Summer M D, Smith G E. A manual of methods for baculovirus vectors and insect cell culture priducedures. Texas Agricultural Experiment station Bulletin.1987,1555.
[111] Maeda S, Kawai T, Obiata M, et al. Production of human alpha-interferon in silkworm using a baculovirus vector[J]. Nature,1985,315:592-594.
[112] Matsuura Y, Possee R D, Bishop D H. Expression of the S-coded genes of lymphocytic choriomeningitis arenavirus using a baclovirus vector[J]. Journal of General Virology,1986, 67: 1515-1529.
[113] Matsuura Y, Possee R D. Bishop D H. Baculovirus expression vectors: The requirement for high level expression of proteins, including glycoprotein[J]. Journal of General Virology, 1987,68:1232-1250.
[114] Lanford R E, Luckow V, Kennedy R C , et al. Expression and characterization of hepatitis B virus surface antigen polypeptides in insect cells with a baculovirus expression system[J]. Journal of Virology, 1989, 63:1549-1557.
[115] Gomi S, Zhou E Z, et al. Delection analysis of eighteen late gene expression factor gene homologues of baculovirus BnNPV[J].Virology,1997,230:35-47.
[116] Manji G A. Baclovirus inhibitor of apoptosis functions at or upstream of the apoptotic suppressor p35 to prevent programmed cell death [J]. Virol,1997,71: 4509-4516.
[117] kihiro U. Improvement of baculovirus expression system(A).prospects for the Development of Insect Factories(M).Japan: Institute of Insect and Animal Science national Institute of Agrobiological Science,2001.57-63.
[118] Muneta Y. Production and utilization of pig cytokines using baculovirus insect expression systems(A).Prospects for the Development of Insect Factories(M).Japan: Institute of Insect and Animal Sciences National Institute of Agrobiological Science,2001,25-33.
[119] Ohura M. The 5th NISES/COE International symposium: Research for the utilization of
insect properties-prospect for the development of new materials?. Tsukuba, Japan.2000.
[120] Ellis A E.Meeting the requirements for delayed release of oral vaccines for fish[J].Journal of Applied Ichthyology,1998,14:149-152.
[121] Inceoglu A B, Kamita S G, Hinton A C, et al. Recombinant baculoviruses for insect control[J].Pest Management Science,2001,57:981-987.
[122] Flores E F, et al. Swine and ruminant pestiviruses require the same cellular factors to enter bovine cells[J]. J Gen Virol, 1996, 77:1295-1303.
[123] Van Ri jn P A,Van gennip H G P, Meijer E J, et al .Epitope mapping of envelop glyprotein E1 of hog cholera virus strain Brescia[J]. Journal of general virology,1993,74:2053-2060.
[124] Kozak M. The scanning model for translation: An update[J]. Jouranal of Cell Biology,1989,108(2):229-241.
[125] Luckow,Y. A.and Summer, M D. Singnals important for high-lever expression of foreign gene in Autographa californica nuclear polyhedrosis virus expression vector[J].Virology,1988,167:56-71.
[126 ]Hammond J M, Mccoy R J, Jansen E S ,et al. Vaccination with a single dose of a recombinant porcine adenovirus expressing the classical swine fever virus gp55(E2) gene protects pigs against classical swine fever[J].Vaccine,2000,18(1-12):1040-1050.