猪瘟病毒HL-LY地方株E2基因在毕赤酵母中的表达及高效表达条件的探索
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摘要
猪瘟是危害养猪业的主要传染病，为了得到大量的猪瘟病毒特异性诊断抗原，并试图在真核细胞中大量表达猪瘟病毒特有的免疫保护性抗原，即E2囊膜糖蛋白，为进一步制备猪瘟亚单位疫苗有效预防本病奠定基础。本试验首次克隆了我国地方分离株HL-LY E2囊膜糖蛋白基因，将该基因整合到毕赤酵母表达系统，使该基因获得了高效表达，并在此基础上探索了不同外界条件对表达量的影响，主要结果如下：
根据基因库已发表的猪瘟病毒E2基因序列及毕赤酵母表达载体pPIC9的多克隆位点序列，利用Oligo和Primer5.0等软件设计并合成一对引物，以提取猪瘟病毒HL-LY株总RNA为模板，应用RT-PCR技术，扩增得到E2基因。将该基因片段先克隆到pMD18-T载体上，并进行了酶切、PCR鉴定和测序分析，阳性重组质粒命名为T-E2。运用DNAMAN软件将测序结果与国内外已发表的猪瘟病毒Alfort株，Brescia株，SHIMEN株，C株，C-V-LZ株，GS-LT株和GS-LX株的E2基因全序列和E2基因主要抗原区序列(23bp～545bp)的核苷酸和氨基酸序列进行了同源性比较，E2基因全序列核苷酸的同源性分别为：99.20％，95.44％，96.51％，95.53％，89.12％，78.66％，78.23％；主要抗原区核苷酸的同源性分别为：98.90％，95.05％，96.70％，96.15％，95.42％，82.42％，83.15％。E2基因全序列氨基酸的同源性分别为：98.93％，95.98％，97.32％，95.71％，89.54％，83.16％，83.42％；主要抗原区氨基酸的同源性分别为：98.35％，95.05％，97.25％，95.60％，95.05％，87.36％，88.46％。结果表明E2基因主要抗原区和全序列均具有较高的保守性，与国内外代表毒株Alfort株，Brescia株，SHIMEN株，C株同源性较高，而与国内其他毒株C-V-LZ株，GS-LT株，GS-LX株同源性较低，说明我国部分地区猪瘟病毒存在E2基因突变株。
将重组质粒T-E2的E2基因酶切后，亚克隆到表达载体pPIC9上，经酶切和PCR鉴定，命名为pPIC9-E2。重组表达载体pPIC9-E2经Sal Ⅰ酶切线性化后，电转化整合到毕赤酵母GS115基因组上，经PCR鉴定和PCR产物测序分析，阳性转化子命名为GS115-pPIC9-E2。
将GS115-pPIC9-E2在30℃条件下振荡培养，以1％的甲醇诱导，猪瘟病毒E2融合蛋白获得了表达，表达量为0.34g／L。表达产物经SDS-PAGE和Western-blot和Dot-ELISA分析，确定其表达的融合蛋白分子量大小为50-54ku，且具有天然蛋白的抗原特异性。
通过对表达条件如pH值、通气量、诱导甲醇终浓度、培养基的选择等条件的探索，可确定猪瘟病毒HL-LY株E2基因表达的理想条件为：最适pH值在4.0～6.0之间，最适培养基为MM培养基，最佳诱导的甲醇终浓度为1％，加大通气量时表达量较好。
Classical Swine Fever(CSF) is one of major contagious diseases in pig breeding industry. In order to gain massive diagnostic antigen CSFV, and large scale of CSFV specific major protective antigen protein(E2 envelope glycoprotein) in eucaryotic cells, which are prepared into subunit vaccine for effective prevention against CSF. E2 gene from CSFV HL-LY isolate was cloned and expressed in Pichia Pastoris expression system, moreover, conditions were explorerd for expressing the E2 envelope glycoprotein. The results are showed as follows.
According to CSFV's E2 gene sequence published in GenBank and the sequence of P.Pastoris expression vector pPIC9's Multiple Cloning Site(MCS), a pair of primers were designed with Oligo and PrimerS.O softwares. E2 gene was amplified by RT-PCR from HL-LY isolate. The product of RT-PCR was cloned to pMD18-T vector, and identified by restriction endonuclease, PCR and sequencing, which was named as T-E2.The E2 gene's sequence was analyzed by homology comparing with published E2 gene of CSFV Alfort strain, Brescia strain, SHIMEN strain, C strain, C-V-LZ strain, GS-LT strain and GS-LX strain.The homology of E2 gene complete sequence shares 99.20%, 95.44%, 96.51%, 95.53%, 89.12%, 78.66% and 78.23% in nucleotide respectively and shares 98.93%, 95.98%, 97.32%, 95.71%, 89.54%, 83.16% and 83.42% identities in amino acid sequence respectively.The major antigenic region of the sequence was also analyzed by homology comparing, which shares 98.90%, 95.05%, 96.70%, 96.15%, 95.42%, 82.42%, and 83.15% in nucleotide respectively and shares 98.35%, 95.05%, 97.25%, 95.60%, 95.05%, 87.36% and 88.46% identities in amino acid sequence respectively.The result shows that E2 gene is higher conservative, but there exist CSFV E2 gene mutation strains in China according to its lower homologies with C-V-LZ , GS-LT and GS-LT strains which were isolated from the different regions in China.
E2 gene was cut with restriction endonuclease from the recombination plasmid T-E2, and subcloned into the expression vector pPIC9's MCS, which was identified by enzyme digestion and PCR amplification. The recombination expression vector, pPIC9-E2, was linearized by Sal I and electroporated into P.Pastoris GS115. Recombinant P.Pastoris GS115, designated GS115-pPIC9-E2, was identified by PCR and the product of the PCR was analyzed by sequencing before its expression for CSFV E2 protein.
Expression of CSFV E2 fusion protein was followed in Recombinant P.Pastoris GS115-pPIC9-E2 with the induction of 1% methanol at 30 C, shaking at 200rpm. A strong protein band of molecular mass estimation 50-54ku with SDS-PAGE was seen. This protein was detected on Western blots and Dot-ELISA probed with anti-CSFV serum, but P.Pastoris GS115 without recombinant E2 showed no protein in this region. It was estimated that the
yield of the E2 fusion protein in culture supernatant of Recombinant P.Pastoris could be reached 0.34g/L.
The optimal expression conditions were explored for the pH, aeration rate, final concentration of methanol and kinds of growth media. We conclude that the higher quantity of E2 protein can be gained at pH4.0-6.0, 1% methanol as the final concentration, MM growth media and high aeration rate.
Candidate:Man Chaolai Speciality:Preventive Veterinary Science Supervisor:Prof. Li Yijing
根据基因库已发表的猪瘟病毒E2基因序列及毕赤酵母表达载体pPIC9的多克隆位点序列，利用Oligo和Primer5.0等软件设计并合成一对引物，以提取猪瘟病毒HL-LY株总RNA为模板，应用RT-PCR技术，扩增得到E2基因。将该基因片段先克隆到pMD18-T载体上，并进行了酶切、PCR鉴定和测序分析，阳性重组质粒命名为T-E2。运用DNAMAN软件将测序结果与国内外已发表的猪瘟病毒Alfort株，Brescia株，SHIMEN株，C株，C-V-LZ株，GS-LT株和GS-LX株的E2基因全序列和E2基因主要抗原区序列(23bp～545bp)的核苷酸和氨基酸序列进行了同源性比较，E2基因全序列核苷酸的同源性分别为：99.20％，95.44％，96.51％，95.53％，89.12％，78.66％，78.23％；主要抗原区核苷酸的同源性分别为：98.90％，95.05％，96.70％，96.15％，95.42％，82.42％，83.15％。E2基因全序列氨基酸的同源性分别为：98.93％，95.98％，97.32％，95.71％，89.54％，83.16％，83.42％；主要抗原区氨基酸的同源性分别为：98.35％，95.05％，97.25％，95.60％，95.05％，87.36％，88.46％。结果表明E2基因主要抗原区和全序列均具有较高的保守性，与国内外代表毒株Alfort株，Brescia株，SHIMEN株，C株同源性较高，而与国内其他毒株C-V-LZ株，GS-LT株，GS-LX株同源性较低，说明我国部分地区猪瘟病毒存在E2基因突变株。
将重组质粒T-E2的E2基因酶切后，亚克隆到表达载体pPIC9上，经酶切和PCR鉴定，命名为pPIC9-E2。重组表达载体pPIC9-E2经Sal Ⅰ酶切线性化后，电转化整合到毕赤酵母GS115基因组上，经PCR鉴定和PCR产物测序分析，阳性转化子命名为GS115-pPIC9-E2。
将GS115-pPIC9-E2在30℃条件下振荡培养，以1％的甲醇诱导，猪瘟病毒E2融合蛋白获得了表达，表达量为0.34g／L。表达产物经SDS-PAGE和Western-blot和Dot-ELISA分析，确定其表达的融合蛋白分子量大小为50-54ku，且具有天然蛋白的抗原特异性。
通过对表达条件如pH值、通气量、诱导甲醇终浓度、培养基的选择等条件的探索，可确定猪瘟病毒HL-LY株E2基因表达的理想条件为：最适pH值在4.0～6.0之间，最适培养基为MM培养基，最佳诱导的甲醇终浓度为1％，加大通气量时表达量较好。
Classical Swine Fever(CSF) is one of major contagious diseases in pig breeding industry. In order to gain massive diagnostic antigen CSFV, and large scale of CSFV specific major protective antigen protein(E2 envelope glycoprotein) in eucaryotic cells, which are prepared into subunit vaccine for effective prevention against CSF. E2 gene from CSFV HL-LY isolate was cloned and expressed in Pichia Pastoris expression system, moreover, conditions were explorerd for expressing the E2 envelope glycoprotein. The results are showed as follows.
According to CSFV's E2 gene sequence published in GenBank and the sequence of P.Pastoris expression vector pPIC9's Multiple Cloning Site(MCS), a pair of primers were designed with Oligo and PrimerS.O softwares. E2 gene was amplified by RT-PCR from HL-LY isolate. The product of RT-PCR was cloned to pMD18-T vector, and identified by restriction endonuclease, PCR and sequencing, which was named as T-E2.The E2 gene's sequence was analyzed by homology comparing with published E2 gene of CSFV Alfort strain, Brescia strain, SHIMEN strain, C strain, C-V-LZ strain, GS-LT strain and GS-LX strain.The homology of E2 gene complete sequence shares 99.20%, 95.44%, 96.51%, 95.53%, 89.12%, 78.66% and 78.23% in nucleotide respectively and shares 98.93%, 95.98%, 97.32%, 95.71%, 89.54%, 83.16% and 83.42% identities in amino acid sequence respectively.The major antigenic region of the sequence was also analyzed by homology comparing, which shares 98.90%, 95.05%, 96.70%, 96.15%, 95.42%, 82.42%, and 83.15% in nucleotide respectively and shares 98.35%, 95.05%, 97.25%, 95.60%, 95.05%, 87.36% and 88.46% identities in amino acid sequence respectively.The result shows that E2 gene is higher conservative, but there exist CSFV E2 gene mutation strains in China according to its lower homologies with C-V-LZ , GS-LT and GS-LT strains which were isolated from the different regions in China.
E2 gene was cut with restriction endonuclease from the recombination plasmid T-E2, and subcloned into the expression vector pPIC9's MCS, which was identified by enzyme digestion and PCR amplification. The recombination expression vector, pPIC9-E2, was linearized by Sal I and electroporated into P.Pastoris GS115. Recombinant P.Pastoris GS115, designated GS115-pPIC9-E2, was identified by PCR and the product of the PCR was analyzed by sequencing before its expression for CSFV E2 protein.
Expression of CSFV E2 fusion protein was followed in Recombinant P.Pastoris GS115-pPIC9-E2 with the induction of 1% methanol at 30 C, shaking at 200rpm. A strong protein band of molecular mass estimation 50-54ku with SDS-PAGE was seen. This protein was detected on Western blots and Dot-ELISA probed with anti-CSFV serum, but P.Pastoris GS115 without recombinant E2 showed no protein in this region. It was estimated that the
yield of the E2 fusion protein in culture supernatant of Recombinant P.Pastoris could be reached 0.34g/L.
The optimal expression conditions were explored for the pH, aeration rate, final concentration of methanol and kinds of growth media. We conclude that the higher quantity of E2 protein can be gained at pH4.0-6.0, 1% methanol as the final concentration, MM growth media and high aeration rate.
Candidate:Man Chaolai Speciality:Preventive Veterinary Science Supervisor:Prof. Li Yijing
引文
1 B．E．斯特劳S．D．阿莱尔等猪病学中国农业大学出版社2000．9
2 韩雪清、刘湘涛等猪瘟病毒及其猪瘟疫苗研究进展动物医学进展2000(2)：1-6
3 蒋太交 吉鑫松等 在甲醇酵母Pichia Pastoris胞内表达有活性的辣根过氧化物酶中国生物化学与分子生物学报2000．2：206—209
4 李红卫 涂长春等 猪瘟病毒保护性抗原E2基因的克隆及在原核细胞中的表达畜牧兽医学报1999 30(2)，146～152
5 刘湘涛、赵启祖、李忠润等 猪瘟病毒(HCV)的研究进展和存在问题[J]，兰州大学学报(自然科学版)1995，3：157—161
6 王家富 张楚瑜等 猪瘟病毒石门株与兔化弱毒株E0糖蛋白基因的克隆与序列分析微生物学报2000，1：32～37
7 王琴 王在时等 12株猪瘟病毒E2基因主要抗原区域的序列差别分析微生物学报2000，6：61
8 王镇、丁孝明 猪瘟病毒致病机理及防治的研究进展畜牧兽医学报1998，29(5)：449-454
9 阎锡蕴 汤健 黄宇鸿 人血管内皮细胞生长因子在毕赤酵母中的表达生物工程学报2000．4：531—533．
10 颜春洪 李春海等 人基质金属蛋白酶—9在酵母Pichia Pastoris中的表达中国生物化学与分子生物学报2000．2：194—199
11 杨剑莹 惠静毅等 带有Pres的重组乙肝表面抗原在毕赤酵母中的表达生物化学与生物物理学报2000．2：139—143
12 杨剑莹 金菁等 毕赤酵母表达的重组乙肝表面抗原SS1的纯化及性质鉴定生物化学与生物物理学报2000．5：503—508
13 殷震等 动物病毒学 北京：科学技术出版社，1985：448—531
14 余兴龙 涂长春等 猪瘟病毒E2基因真核表达质粒的构建及基因疫苗的研究中国病毒学2000 3：264～270
15 张冬梅 潘卫庆等恶性疟疾裂殖子表面蛋白1合成基因在毕赤酵母中表达生物工程学报2000 6：723—726
16 周鹏程 陈建国等 猪瘟病毒糖蛋白E0基因的克隆及其表达研究微生物学通报2000．(3)165～170
17 Allen D.Leman, Barbara E.Straw. et.al,.DISEASES OF SWINE IOWA STATE University Press/Ames, IOWA; USA. 1992.274-285
18 Andrew M E, Morrissy C J, Lenghaus C, et al.Protection of Pigs Against Classical Swine Fever with DNA-delivered gp55 [J].Vaccine, 2000, 18:1932～1938.
19 Barlak P. Genetic Typing of Classical Swine Fever Virus Isolate from Czech Republic, Vet. Microboil, 2000 Nov. 15(2):59-70
20 BE0 A.Live Attenuated Pseudorabies Virus Expressing Envelop Glyloprotein E1 of Hog
Cholera Virus Protects Swine Against both Pseudorabies and Hog Cholera [J].J Virol, 1991, 65:2761～2765.
21 Brown EA, Zhang H, Ping LH, Lemon SM.Secondary Structure of the 5' Nontranslated Regions of Hepatitis C Virus and Pestivirus Genomic RNAs. Nucleic Acids Res 1992 Oct 11 ;20(19): 5041-5
22 De Smit A J, Bouma A, Van Gennip H G P, et al.Chimeric (marker) C-strain Virus Induce Classical Protection Against Virulent Classical Swine Fever Viruses (CSFV) and Reduce Transmission of CSFV between Vaccinated Pig [J]. Vaccine, 2001, 19:1467～1476.
23 De Smit A J, Van Gennip H G P, Miedema G K W, et al. Prevention of Transplacental Transmission of Moderate-virulent Classical Swine Fever Viruses(CSFV) after a Single or Double Vaccination with a CSF E2 Subunit vaccine [J].Vet Quart, 2000, 22:150～153.
24 Deng R, Brock K V.5' and 3' Untranslated Regious of Pestivirus Genome:Primery and Sondary Structure Analyse.Nucleic Acids Res, 1993, 21:1949-1957
25 Edwards S, Moennig V, Wensvoort G.The Development of an International Reference Panel of Monoclonal Antibodies for the Differentiation of Hog Cholera Virus from other Pestiviruses. Vet Microbiol 1991 Oct;29(2): 101-8
26 Francko R. I .B, Faguet C .M, Knuolson, D. L and Brown. F. (Eds,) Arch Vird Suppl 2, 223—233(1991).
27 Hahn J.Construction of Recombinant Swinepox Viruses and Expression of the Classical Swine Fever Virus E2 Protein [J].J Virol Methods, 2001, 93(1-2):49～56.
28 Hammond J M, Jansen E S, Morrissy C J, et al.A Primeboost Vaaccination Strategy Using Naked DNA Followed by Recombinant Porcine Adenovirus Protects Pigs from Classical Swine Fever [J].Vet Microbiol, 2001, 80(2):101～119.
29 Hulst MM, van Gennip HG, Moormann RJ.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 E(ms). J Virol 2000 Oct;74(20):9553-61
30 Huslt M M, Westra D F, Wenvoort G, et al. Glycoprotein E2 of Hog Cholera Virus Espressed in Insetells Protects Swine from Hog Cholera [J].J Virol, 1993, 67:5435～5442.
31 Khromykh AA, Westaway EG.Completion of Kunjin Virus RNA Sequence and Recovery of an Infectious RNA Transcribed from Stably Cloned Full-length cDNA. J Virol 1994 Jul;68(7):4580-8
32 Konig M, Lengsfeld T, Pauly T, Stark R, Thiel HJ.Classieal Swine Fever Virus: Independent Induction of Protective Immunity by two Structural Glycoproteins. J Virol 1995 Oct;69(10):6479-86
33 Le SY, Sonenberg N, Maizel JV Jr. Unusual Folding Regions and Ribosome Landing Pad within Hepatitis C Virus and Pestivirus RNAs. Gene 1995 Mar 10; 154(2): 137-43
34 Lin M, Lin F, Mallory M, Clavijo A.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 Virol 2000 Dec;74(24): 11619-25
35 Meyers G, Rumenapf T, Thiel HJ.Molecular Cloning and Nucleotide Sequence of the Genome of Hog Cholera Virus. Virology 1989 Aug;171(2):555-67
36 Meyers G, Saalmuller A, Buttner M.Mutations Abrogating the RNase Activity in Glycoprotein E(rns) of the Pestivirus Classical Swine Fever Virus Lead to Virus Attenuation. J Virol 1999 Dec;73(12): 10224-35
37 Meyers G, Thiel H J, Molecular Characterization of Pestiviruses[J], Adv Vitrus Res, 1996, 47:53-118
38 Moennig, v and Plangemann, P.G.W, adv, Virus Res. 41, 53—98(1992)
39 Moormann R J M, Van Gen H G P, Miedema G K W, et al. Infectious RNA Transcribed from on Engineered Full-length cDNA Template of the Genome of a Pestivirus .J Virol, 1996, 2:736-770
40 Moormann R J M, Van Gennip H G P, Miedema G K W, et al. Infection RNA Transcribed from an Engineered Full-length cDNA Template of the Genome of a Pestivirus [J]. J Virol, 1996, 70:763-770.
41 Moormann R J, Bouma A.Development of a Classical Swine Fever Subunit Marker Vaccine and Companiondiagnostie Test [J].Vet Microbiol, 2000, 73(2～3):209～219.
42 Moormann R J, van Gennip HG, Miedema GK, Hulst MM, van Rijn PA.Infectious RNA Transcribed from an Engineered Full-length cDNA Template of the Genome of a Pestivirus. J Virol 1996 Feb;70(2):763-70
43 Moormann R J, Warmerdam PA, van der Meer B, Hulst MM.Nucleotide Sequence of Hog Cholera Virus RNA: Properties of the Polyprotein Encoded by the Open Reading Frame Spanning the Viral Genomic RNA. Vet Microbiol 1990 Jun;23(1-4): 185-91
44 Moormann R J, Warmerdam PA, van der Meer B, Schaaper WM, Wensvoort G, Hulst M.Molecular Cloning and Nucleotide Sequence of Hog Cholera vVirus Strain Brescia and Mapping of the Genomic Region Encoding Envelope Protein E1. Virology 1990 Jul; 177(1): 184-98
45 Moser C, Stettler P, Tratschin JD, Hofmann MA.Cytopathogenic and Noncytopathogenic RNA Replicons of Classical Swine Fever Virus. J Virol 1999 Sep;73(9):7787-94
46 Owings P, Ibata G, Needham J, Paton D. Classical Swine Fever Virus Diversity and Evolution. J Gen Virol. 1996 Jun:77(Pt 6): 1311-1321
47 Paton DJ, Lowings JP, Barrett AD.Epitope Mapping of the gp53 Envelope Protein of Bovine Viral Diarrhea Virus. Virology 1992 Oct; 190(2):763-72
48 Pauly T, et al. Classical Swine Fever Viruses-specific Cytotoxie T LympHocytes and Identification of a T Cell Epitope.J.Gen Virol, 1995, 76:3039～3049.
49 Peeters B, Bienkowska-Szewczyk K, Hulst M, Gielkens A, Kimman T. Biologically Safe, Non-transmissible Pseudorahies Vires Vector Vaccine Protects Pigs Against Both Aujeszky's Disease and Classical Swine Fever. J Gen Virol. 1997 Dec; 78 (Pt 12):3311-5.
50 Renard A, Guiot C, Schmetz D, Dagenais L, Pastoret PP, Dina D, Martial JA.Molecular Cloning of Bovine Viral Diarrhea Viral Sequences. DNA 1985 Dec;4(6):429-38
51 Rijnbrand R, Straaten T V D, Rijn P A V, et al.Intetnal Entry of Ribosome is Derected by the 5' ding Region of Classical Swine Fever Viruses and is Dependent on the Prence of an RNA Pseudoknot Upstream of the Initiation Codon [J].J Virol, 1997,71(1):451～457.
52 Rumenapf T, Unger G, Strauss JH, Thiel HJ.Processing of the Envelope Glycoproteins of Pestiviruses. J Virol 1993 Jun;67(6):3288-94
53 Rumenupf T, Stark R, Meyers G, et al. Structural Proteins of Hog Cholera Virus Expressed by Vaccine Virus:Further Characterization and Induction of Protective Immunity [J].J Virol, 1991, 65:589～597.
54 Stark R, Meyers G, Rumenapf T, Thiel HJ Processing of Pestivirus Polyprotein: Cleavage Site Between Autoprotease and Nucleocapsid Protein of Classical Swine Fever Virus. J Virol 1993 Dec;67(12):7088-95
55 Stark R, Rumenapf T, Meyers G, Thiel HJ.Genomic Localization of Hog Cholera Virus Glycoproteins. Virology 1990 Jan; 174(1):286-9
56 Tratschin J D, 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.
57 Van Gennip H G P, Van Rijn P A, Widjojoatmodjo M N, et al. Chimeric Classical Swine Fever Viruses Containing Envelope Protein E0 or E2 of Bovine Viral Diarrhea Virus Protect Pigs Against Challenge with CSFV and Induce a Distinguishable Antibody Response [J]. Vaccine, 2001, 19:447～459.
58 Van Rijn P A, Miedema G K W, Wensvoort G, et al.Antigenic Structure of Envelope Glycoprotein E1 of Hog Cholera Virus [J].J Virol, 1994, 68:3934～3942.
59 Van Rijn PA, Miedema GK, Wensvoort G, van Gennip HG, Moormann RJ.Antigenic Etmcture of Envelope Glycoprotein E1 of Hog Cholera Virus. J Virol 1994 Jun;68(6):3934-42
60 Van Rijn PA, van Gennip HG, de Meijer EJ, Moormann RJ.Epitope Mapping of Envelope Glycoprotein E1 of Hog Cholera Virus Strain Brescia. J Gen Virol 1993 Oct;74 (Pt 10):2053-60
61 Van Zijl M, Wensvoort G, De Kluyver E, et al. Live Attenuated Pseudorabies Virus Expressing Envelope Glycoprotein E1 of Hog Cholera Virus Protects Swine Against Both Pseudorabies and Hog Cholera [J].J Viroh 1991, 65:2761～2765.
62 Vanderhallen H, Mittelholzer C, Hofmann MA, Koenen F, Mittelhozer C.Classical Swine
Fever Virus is Genetically Stable in Vitro and in Vivo. Arch Virol 1999; 144(9): 1669-77
63 Weiland E, Stark R, Haas B, Rumenapf T, Meyers G, Thiel HJ.Pestivirus Glycoprotein Which Induces Neutralizing Antibodies Forms Part of a Disulfide-linked Heterodimer. J Virol 1990 Aug;64(8):3563-9
64 Wengler G.Family Flaviviridae .In Franchi R I B et al (eds), classification and nomenclature of viruses, Fifth report of the International Committee on Taxonomy of Virus, Berlin, Spriinger Verlaf, 1991, 223～233
65 Wensvoort G, Terpstra C, de Kluijver EP, Kragten C, Warnaar JC.Antigenic Differentiation of Pestivirus Strains with Monoclonal Antibodies Against Hog Cholera Virus. Vet Microbiol 1989 Nov;21(1):9-20
66 Windisch JM, Schneider R, Stark R, Weiland E, Meyers G, Thiel HJ.RNase of Classical Swine Fever Virus: Biochemical Characterization and Inhibition by Virus-neutralizing Monoclonal Antibodies. J Virol 1996 Jan;70(1):352-8
67 Yu M, Wang L F, Shiell B J, et al. Fine Mapping of a C-terminal Linear Epitope Highly Conserved among the Major Enevelope Glycoprotein E2(gp51 or gp54) of Different Pestiviruses [J].Virology, 1996, 222(1):289～292.
68 YU X. DNA-mediated Protection against Classical Swine Fever Virus, Vaccine 2001, Jan 8.19(11-12):1520-1525
2 韩雪清、刘湘涛等猪瘟病毒及其猪瘟疫苗研究进展动物医学进展2000(2)：1-6
3 蒋太交 吉鑫松等 在甲醇酵母Pichia Pastoris胞内表达有活性的辣根过氧化物酶中国生物化学与分子生物学报2000．2：206—209
4 李红卫 涂长春等 猪瘟病毒保护性抗原E2基因的克隆及在原核细胞中的表达畜牧兽医学报1999 30(2)，146～152
5 刘湘涛、赵启祖、李忠润等 猪瘟病毒(HCV)的研究进展和存在问题[J]，兰州大学学报(自然科学版)1995，3：157—161
6 王家富 张楚瑜等 猪瘟病毒石门株与兔化弱毒株E0糖蛋白基因的克隆与序列分析微生物学报2000，1：32～37
7 王琴 王在时等 12株猪瘟病毒E2基因主要抗原区域的序列差别分析微生物学报2000，6：61
8 王镇、丁孝明 猪瘟病毒致病机理及防治的研究进展畜牧兽医学报1998，29(5)：449-454
9 阎锡蕴 汤健 黄宇鸿 人血管内皮细胞生长因子在毕赤酵母中的表达生物工程学报2000．4：531—533．
10 颜春洪 李春海等 人基质金属蛋白酶—9在酵母Pichia Pastoris中的表达中国生物化学与分子生物学报2000．2：194—199
11 杨剑莹 惠静毅等 带有Pres的重组乙肝表面抗原在毕赤酵母中的表达生物化学与生物物理学报2000．2：139—143
12 杨剑莹 金菁等 毕赤酵母表达的重组乙肝表面抗原SS1的纯化及性质鉴定生物化学与生物物理学报2000．5：503—508
13 殷震等 动物病毒学 北京：科学技术出版社，1985：448—531
14 余兴龙 涂长春等 猪瘟病毒E2基因真核表达质粒的构建及基因疫苗的研究中国病毒学2000 3：264～270
15 张冬梅 潘卫庆等恶性疟疾裂殖子表面蛋白1合成基因在毕赤酵母中表达生物工程学报2000 6：723—726
16 周鹏程 陈建国等 猪瘟病毒糖蛋白E0基因的克隆及其表达研究微生物学通报2000．(3)165～170
17 Allen D.Leman, Barbara E.Straw. et.al,.DISEASES OF SWINE IOWA STATE University Press/Ames, IOWA; USA. 1992.274-285
18 Andrew M E, Morrissy C J, Lenghaus C, et al.Protection of Pigs Against Classical Swine Fever with DNA-delivered gp55 [J].Vaccine, 2000, 18:1932～1938.
19 Barlak P. Genetic Typing of Classical Swine Fever Virus Isolate from Czech Republic, Vet. Microboil, 2000 Nov. 15(2):59-70
20 BE0 A.Live Attenuated Pseudorabies Virus Expressing Envelop Glyloprotein E1 of Hog
Cholera Virus Protects Swine Against both Pseudorabies and Hog Cholera [J].J Virol, 1991, 65:2761～2765.
21 Brown EA, Zhang H, Ping LH, Lemon SM.Secondary Structure of the 5' Nontranslated Regions of Hepatitis C Virus and Pestivirus Genomic RNAs. Nucleic Acids Res 1992 Oct 11 ;20(19): 5041-5
22 De Smit A J, Bouma A, Van Gennip H G P, et al.Chimeric (marker) C-strain Virus Induce Classical Protection Against Virulent Classical Swine Fever Viruses (CSFV) and Reduce Transmission of CSFV between Vaccinated Pig [J]. Vaccine, 2001, 19:1467～1476.
23 De Smit A J, Van Gennip H G P, Miedema G K W, et al. Prevention of Transplacental Transmission of Moderate-virulent Classical Swine Fever Viruses(CSFV) after a Single or Double Vaccination with a CSF E2 Subunit vaccine [J].Vet Quart, 2000, 22:150～153.
24 Deng R, Brock K V.5' and 3' Untranslated Regious of Pestivirus Genome:Primery and Sondary Structure Analyse.Nucleic Acids Res, 1993, 21:1949-1957
25 Edwards S, Moennig V, Wensvoort G.The Development of an International Reference Panel of Monoclonal Antibodies for the Differentiation of Hog Cholera Virus from other Pestiviruses. Vet Microbiol 1991 Oct;29(2): 101-8
26 Francko R. I .B, Faguet C .M, Knuolson, D. L and Brown. F. (Eds,) Arch Vird Suppl 2, 223—233(1991).
27 Hahn J.Construction of Recombinant Swinepox Viruses and Expression of the Classical Swine Fever Virus E2 Protein [J].J Virol Methods, 2001, 93(1-2):49～56.
28 Hammond J M, Jansen E S, Morrissy C J, et al.A Primeboost Vaaccination Strategy Using Naked DNA Followed by Recombinant Porcine Adenovirus Protects Pigs from Classical Swine Fever [J].Vet Microbiol, 2001, 80(2):101～119.
29 Hulst MM, van Gennip HG, Moormann RJ.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 E(ms). J Virol 2000 Oct;74(20):9553-61
30 Huslt M M, Westra D F, Wenvoort G, et al. Glycoprotein E2 of Hog Cholera Virus Espressed in Insetells Protects Swine from Hog Cholera [J].J Virol, 1993, 67:5435～5442.
31 Khromykh AA, Westaway EG.Completion of Kunjin Virus RNA Sequence and Recovery of an Infectious RNA Transcribed from Stably Cloned Full-length cDNA. J Virol 1994 Jul;68(7):4580-8
32 Konig M, Lengsfeld T, Pauly T, Stark R, Thiel HJ.Classieal Swine Fever Virus: Independent Induction of Protective Immunity by two Structural Glycoproteins. J Virol 1995 Oct;69(10):6479-86
33 Le SY, Sonenberg N, Maizel JV Jr. Unusual Folding Regions and Ribosome Landing Pad within Hepatitis C Virus and Pestivirus RNAs. Gene 1995 Mar 10; 154(2): 137-43
34 Lin M, Lin F, Mallory M, Clavijo A.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 Virol 2000 Dec;74(24): 11619-25
35 Meyers G, Rumenapf T, Thiel HJ.Molecular Cloning and Nucleotide Sequence of the Genome of Hog Cholera Virus. Virology 1989 Aug;171(2):555-67
36 Meyers G, Saalmuller A, Buttner M.Mutations Abrogating the RNase Activity in Glycoprotein E(rns) of the Pestivirus Classical Swine Fever Virus Lead to Virus Attenuation. J Virol 1999 Dec;73(12): 10224-35
37 Meyers G, Thiel H J, Molecular Characterization of Pestiviruses[J], Adv Vitrus Res, 1996, 47:53-118
38 Moennig, v and Plangemann, P.G.W, adv, Virus Res. 41, 53—98(1992)
39 Moormann R J M, Van Gen H G P, Miedema G K W, et al. Infectious RNA Transcribed from on Engineered Full-length cDNA Template of the Genome of a Pestivirus .J Virol, 1996, 2:736-770
40 Moormann R J M, Van Gennip H G P, Miedema G K W, et al. Infection RNA Transcribed from an Engineered Full-length cDNA Template of the Genome of a Pestivirus [J]. J Virol, 1996, 70:763-770.
41 Moormann R J, Bouma A.Development of a Classical Swine Fever Subunit Marker Vaccine and Companiondiagnostie Test [J].Vet Microbiol, 2000, 73(2～3):209～219.
42 Moormann R J, van Gennip HG, Miedema GK, Hulst MM, van Rijn PA.Infectious RNA Transcribed from an Engineered Full-length cDNA Template of the Genome of a Pestivirus. J Virol 1996 Feb;70(2):763-70
43 Moormann R J, Warmerdam PA, van der Meer B, Hulst MM.Nucleotide Sequence of Hog Cholera Virus RNA: Properties of the Polyprotein Encoded by the Open Reading Frame Spanning the Viral Genomic RNA. Vet Microbiol 1990 Jun;23(1-4): 185-91
44 Moormann R J, Warmerdam PA, van der Meer B, Schaaper WM, Wensvoort G, Hulst M.Molecular Cloning and Nucleotide Sequence of Hog Cholera vVirus Strain Brescia and Mapping of the Genomic Region Encoding Envelope Protein E1. Virology 1990 Jul; 177(1): 184-98
45 Moser C, Stettler P, Tratschin JD, Hofmann MA.Cytopathogenic and Noncytopathogenic RNA Replicons of Classical Swine Fever Virus. J Virol 1999 Sep;73(9):7787-94
46 Owings P, Ibata G, Needham J, Paton D. Classical Swine Fever Virus Diversity and Evolution. J Gen Virol. 1996 Jun:77(Pt 6): 1311-1321
47 Paton DJ, Lowings JP, Barrett AD.Epitope Mapping of the gp53 Envelope Protein of Bovine Viral Diarrhea Virus. Virology 1992 Oct; 190(2):763-72
48 Pauly T, et al. Classical Swine Fever Viruses-specific Cytotoxie T LympHocytes and Identification of a T Cell Epitope.J.Gen Virol, 1995, 76:3039～3049.
49 Peeters B, Bienkowska-Szewczyk K, Hulst M, Gielkens A, Kimman T. Biologically Safe, Non-transmissible Pseudorahies Vires Vector Vaccine Protects Pigs Against Both Aujeszky's Disease and Classical Swine Fever. J Gen Virol. 1997 Dec; 78 (Pt 12):3311-5.
50 Renard A, Guiot C, Schmetz D, Dagenais L, Pastoret PP, Dina D, Martial JA.Molecular Cloning of Bovine Viral Diarrhea Viral Sequences. DNA 1985 Dec;4(6):429-38
51 Rijnbrand R, Straaten T V D, Rijn P A V, et al.Intetnal Entry of Ribosome is Derected by the 5' ding Region of Classical Swine Fever Viruses and is Dependent on the Prence of an RNA Pseudoknot Upstream of the Initiation Codon [J].J Virol, 1997,71(1):451～457.
52 Rumenapf T, Unger G, Strauss JH, Thiel HJ.Processing of the Envelope Glycoproteins of Pestiviruses. J Virol 1993 Jun;67(6):3288-94
53 Rumenupf T, Stark R, Meyers G, et al. Structural Proteins of Hog Cholera Virus Expressed by Vaccine Virus:Further Characterization and Induction of Protective Immunity [J].J Virol, 1991, 65:589～597.
54 Stark R, Meyers G, Rumenapf T, Thiel HJ Processing of Pestivirus Polyprotein: Cleavage Site Between Autoprotease and Nucleocapsid Protein of Classical Swine Fever Virus. J Virol 1993 Dec;67(12):7088-95
55 Stark R, Rumenapf T, Meyers G, Thiel HJ.Genomic Localization of Hog Cholera Virus Glycoproteins. Virology 1990 Jan; 174(1):286-9
56 Tratschin J D, 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.
57 Van Gennip H G P, Van Rijn P A, Widjojoatmodjo M N, et al. Chimeric Classical Swine Fever Viruses Containing Envelope Protein E0 or E2 of Bovine Viral Diarrhea Virus Protect Pigs Against Challenge with CSFV and Induce a Distinguishable Antibody Response [J]. Vaccine, 2001, 19:447～459.
58 Van Rijn P A, Miedema G K W, Wensvoort G, et al.Antigenic Structure of Envelope Glycoprotein E1 of Hog Cholera Virus [J].J Virol, 1994, 68:3934～3942.
59 Van Rijn PA, Miedema GK, Wensvoort G, van Gennip HG, Moormann RJ.Antigenic Etmcture of Envelope Glycoprotein E1 of Hog Cholera Virus. J Virol 1994 Jun;68(6):3934-42
60 Van Rijn PA, van Gennip HG, de Meijer EJ, Moormann RJ.Epitope Mapping of Envelope Glycoprotein E1 of Hog Cholera Virus Strain Brescia. J Gen Virol 1993 Oct;74 (Pt 10):2053-60
61 Van Zijl M, Wensvoort G, De Kluyver E, et al. Live Attenuated Pseudorabies Virus Expressing Envelope Glycoprotein E1 of Hog Cholera Virus Protects Swine Against Both Pseudorabies and Hog Cholera [J].J Viroh 1991, 65:2761～2765.
62 Vanderhallen H, Mittelholzer C, Hofmann MA, Koenen F, Mittelhozer C.Classical Swine
Fever Virus is Genetically Stable in Vitro and in Vivo. Arch Virol 1999; 144(9): 1669-77
63 Weiland E, Stark R, Haas B, Rumenapf T, Meyers G, Thiel HJ.Pestivirus Glycoprotein Which Induces Neutralizing Antibodies Forms Part of a Disulfide-linked Heterodimer. J Virol 1990 Aug;64(8):3563-9
64 Wengler G.Family Flaviviridae .In Franchi R I B et al (eds), classification and nomenclature of viruses, Fifth report of the International Committee on Taxonomy of Virus, Berlin, Spriinger Verlaf, 1991, 223～233
65 Wensvoort G, Terpstra C, de Kluijver EP, Kragten C, Warnaar JC.Antigenic Differentiation of Pestivirus Strains with Monoclonal Antibodies Against Hog Cholera Virus. Vet Microbiol 1989 Nov;21(1):9-20
66 Windisch JM, Schneider R, Stark R, Weiland E, Meyers G, Thiel HJ.RNase of Classical Swine Fever Virus: Biochemical Characterization and Inhibition by Virus-neutralizing Monoclonal Antibodies. J Virol 1996 Jan;70(1):352-8
67 Yu M, Wang L F, Shiell B J, et al. Fine Mapping of a C-terminal Linear Epitope Highly Conserved among the Major Enevelope Glycoprotein E2(gp51 or gp54) of Different Pestiviruses [J].Virology, 1996, 222(1):289～292.
68 YU X. DNA-mediated Protection against Classical Swine Fever Virus, Vaccine 2001, Jan 8.19(11-12):1520-1525
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