猪瘟病毒E2基因部分片段的克隆和原核表达及其抗体制备
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摘要
E2蛋白是猪瘟病毒的跨膜结构糖蛋白,是最易发生变异的一种蛋白,能诱导感染动物产生保护性免疫和猪瘟病毒中和抗体,是一种免疫优势蛋白,其中A1、B、C区对介导中和抗体的产生很重要,用A1和B或A1和C的单抗可见协同中和反应。表达CSFV Brescia株E2蛋白重组伪狂犬病毒以及用亲和层析法从昆虫细胞中的基因工程E2蛋白均能保护免疫猪抵抗猪瘟病毒的感染,可以确定E2蛋白诱导的免疫反应足以保护免疫猪抵抗猪瘟病毒的感染。为制备猪瘟病毒E2蛋白的抗体,研究E2基因表达产物在免疫检测中的应用及E2蛋白的中和性抗原表位,我们进行了猪瘟病毒E2基因部分片段的克隆和原核表达。
首先根据GenBank中登录的猪瘟病毒基因组序列,设计一对引物,利用聚合酶链式反应(Polymerase chain reaction,PCR)技术从本实验室保存的重组质粒(PGEX-4T-1-PE2)中扩增出大小约为622bp的基因;经单酶切鉴定,与已知的猪瘟病毒E2基因含有相同的DraⅠ酶切位点。PCR产物分离纯化后连接到pMD18-T载体,并转化宿主菌DH5α,经菌液PCR筛选出阳性重组克隆PMD18-T-TE2,结果表明本实验成功的克隆到猪瘟病毒TE2基因。
其次,根据所克隆的猪瘟病毒TE2基因序列和原核表达载体PGEX-4T-1多克隆酶切位点设计另一对引物,应用PCR技术从重组质粒PMD18-T-TE2中扩增出长约622bp的猪瘟病毒TE2基因,编码包括CSFV E2囊膜糖蛋白主要抗原区域A、B、C和D。再将扩增的猪瘟病毒TE2基因克隆到原核表达载体PGEX-4T-1中,经质粒PCR和EcoRⅠ、SalⅠ双酶切鉴定,筛选出阳性重组克隆,构建原核表达重组质粒PGEX-4T-1-TE2。然后将该重组质粒转化大肠杆菌(EcoliRosetta(DE3)BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)表达出谷胱甘肽-S-转移酶(GST)与TE2的融合蛋白GST-TE2,融合蛋白的分子量约为47.0KD,与预期结果相符。通过优化原核表达条件,确定了原核表达的最佳诱导时间和诱导剂浓度。对表达蛋白的可溶性进行鉴定,结果表明表达产物GST-TE2融合蛋白以包涵体形式存在。我们用优化的原核表达条件对含有PGEX-4T-1-TE2质粒的Rosetta(DE3)BL21菌进行大量诱导表达,反复冻融和超声方法裂解诱导菌,用十二烷基肌氨酸钠加超声波的方法使包涵体充分溶解,Folin-酚法测定提取的融合蛋白GST-TE2浓度约为1.2mg/ml。应用提取的GST-TE2融合蛋白作为抗原免疫小鼠制备鼠抗猪瘟病毒TE2多克隆抗体。琼脂扩散实验表明制备的多抗具有良好的反应性,ELISA实验表明鼠多抗的有效稀释度可达1:25600,免疫印记(Western-blotting,WB)实验证实鼠抗猪瘟病毒GST-TE2多抗含有抗GST抗体和抗融合蛋白GST-TE2抗体。
综上所述,本研究成功地克隆了猪瘟病毒TE2基因,成功构建了原核表达质粒PGEX-4T-1-TE2并进行了原核表达,提取了融合蛋白GST-TE2,并且制备了效价高、反应性强的鼠源抗猪瘟病毒GST-TE2多克隆抗体,这些为进一步研究猪瘟病毒E2蛋白免疫学功能及其应用提供了试验材料。
E2 protein is transmembrane glycoprotein of Classical Swine Fever Virus(CSFV), prone to mutation, a kind of immunologic preponderant protein, and can induce infected animals to protective immunity and neutral antibody of CSFV,section A1、B、C is very important for engendering neutral antibody, cooperative neutral reaction is happened when using monoclonal antibody A1 and B or A1 and C. Expression of recombining CSFV Brescia strain E2 gene to Pseudorabies Virus and purification of gene engineering E2 protein by affinity chromatography from insects cells both protect immune pigs from infection of CSFV. In order to prepare antibody against CSFV E2 protein, do research on application of expressed production of E2 gene to immunologic detection and neutral antigen epitopes of E2 protein, partial fragment of CSFV E2 gene was cloned and expressed.
Firstly, specific primers for CSFV E2 gene were designed according to the CSFV genomic sequence registered in GeneBank.Using PCR technology, one gene fragment about 622bp was amplified from a recombinant plasmid PGEX-4T-1- PE2 which was conserved in our experiment. The result of endonuclease Dra I digesting PCR product showed that this gene fragment has the same restriction enzyme site as known CSFV E2 gene. Gene fragments were ligated to pMD18-T vector after purification, the recombinant plasmid pMD18-T-TE2 was transformed into Escherichia coli (E.coli)DH5α,the recombinants were analyzed after they were identified by PCR, subsequently. This suggests that the CSFV TE2 gene has been successfully cloned.
Secondly, gene-specific primers were designed based on the cloned TE2 nucleotide sequences and multi-clone restriction enzyme sites of prokaryotic expressive vector pGEX-4T-l, one gene fragment which encode major antigenic domains of E2(A、B、C、D) about 622bp was amplified from a recombinant plasmid pMD18-T-TE2 by PCR. Then the cDNA fragment which encode major antigenic domains of E2(A、B、C、D) was cloned into pGEX-4T-l,the recombinant plasmid PGEX-4T-1-TE2 was identified by PCR and endonuclease EcoR I +Sal I digestion. The PGEX-4T-1-TE2 was transformed into E.coli Rosetta BL21 DE3 and GST-TE2 fusion protein was induced to express by isopropylthio-β-D-galactoside(IPTG). The MW of the fusion protein was about 47 000 as analyzed by SDS-PAGE.After optimizing prokaryotic expression conditions, we determined the optimum inducement time and concentration of IPTG. The solubility of GST-TE2 fusion protein was identified and the result indicated that expressed GST-TE2 protein existed as inclusion bodies. The recombinant plasmid was induced to express large-scale GST-TE2 fusion protein under the optimal condition, the induced recombinant bacteria was lysed by freeze-thaw and sonication. We obtained the GST-TE2 inclusion body protein, which could be solubilized by sonication after the detergent lauroylsarcosine was added. The concentration of fusion protein GST-TE2 was about 1.2mg/ml by folin-hydroxybenzene.
Finally, using the GST-TE2 fusion protein as antigen, polyclonal antiserum to CSFV E2 was derived from mouse. Results of agar diffusion assay demonstrated that the polyclonal antibody could well react to TE2 protein. ELISA indicated that the immunized rabbits had antibody titers of 1:25600.Western-blotting showed that polyclonal antibody against CSFV GST-TE2 from mouse contain antibody against GST and antibody against GST-TE2 fusion protein.
In conclusion, we have successfully cloned、expressed CSFV TE2 gene, and obtained the polyclonal antibody against CSFV TE2.A11 these provide some experimental materials for the future studies on the immunological function of CSFV E2.
首先根据GenBank中登录的猪瘟病毒基因组序列,设计一对引物,利用聚合酶链式反应(Polymerase chain reaction,PCR)技术从本实验室保存的重组质粒(PGEX-4T-1-PE2)中扩增出大小约为622bp的基因;经单酶切鉴定,与已知的猪瘟病毒E2基因含有相同的DraⅠ酶切位点。PCR产物分离纯化后连接到pMD18-T载体,并转化宿主菌DH5α,经菌液PCR筛选出阳性重组克隆PMD18-T-TE2,结果表明本实验成功的克隆到猪瘟病毒TE2基因。
其次,根据所克隆的猪瘟病毒TE2基因序列和原核表达载体PGEX-4T-1多克隆酶切位点设计另一对引物,应用PCR技术从重组质粒PMD18-T-TE2中扩增出长约622bp的猪瘟病毒TE2基因,编码包括CSFV E2囊膜糖蛋白主要抗原区域A、B、C和D。再将扩增的猪瘟病毒TE2基因克隆到原核表达载体PGEX-4T-1中,经质粒PCR和EcoRⅠ、SalⅠ双酶切鉴定,筛选出阳性重组克隆,构建原核表达重组质粒PGEX-4T-1-TE2。然后将该重组质粒转化大肠杆菌(EcoliRosetta(DE3)BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)表达出谷胱甘肽-S-转移酶(GST)与TE2的融合蛋白GST-TE2,融合蛋白的分子量约为47.0KD,与预期结果相符。通过优化原核表达条件,确定了原核表达的最佳诱导时间和诱导剂浓度。对表达蛋白的可溶性进行鉴定,结果表明表达产物GST-TE2融合蛋白以包涵体形式存在。我们用优化的原核表达条件对含有PGEX-4T-1-TE2质粒的Rosetta(DE3)BL21菌进行大量诱导表达,反复冻融和超声方法裂解诱导菌,用十二烷基肌氨酸钠加超声波的方法使包涵体充分溶解,Folin-酚法测定提取的融合蛋白GST-TE2浓度约为1.2mg/ml。应用提取的GST-TE2融合蛋白作为抗原免疫小鼠制备鼠抗猪瘟病毒TE2多克隆抗体。琼脂扩散实验表明制备的多抗具有良好的反应性,ELISA实验表明鼠多抗的有效稀释度可达1:25600,免疫印记(Western-blotting,WB)实验证实鼠抗猪瘟病毒GST-TE2多抗含有抗GST抗体和抗融合蛋白GST-TE2抗体。
综上所述,本研究成功地克隆了猪瘟病毒TE2基因,成功构建了原核表达质粒PGEX-4T-1-TE2并进行了原核表达,提取了融合蛋白GST-TE2,并且制备了效价高、反应性强的鼠源抗猪瘟病毒GST-TE2多克隆抗体,这些为进一步研究猪瘟病毒E2蛋白免疫学功能及其应用提供了试验材料。
E2 protein is transmembrane glycoprotein of Classical Swine Fever Virus(CSFV), prone to mutation, a kind of immunologic preponderant protein, and can induce infected animals to protective immunity and neutral antibody of CSFV,section A1、B、C is very important for engendering neutral antibody, cooperative neutral reaction is happened when using monoclonal antibody A1 and B or A1 and C. Expression of recombining CSFV Brescia strain E2 gene to Pseudorabies Virus and purification of gene engineering E2 protein by affinity chromatography from insects cells both protect immune pigs from infection of CSFV. In order to prepare antibody against CSFV E2 protein, do research on application of expressed production of E2 gene to immunologic detection and neutral antigen epitopes of E2 protein, partial fragment of CSFV E2 gene was cloned and expressed.
Firstly, specific primers for CSFV E2 gene were designed according to the CSFV genomic sequence registered in GeneBank.Using PCR technology, one gene fragment about 622bp was amplified from a recombinant plasmid PGEX-4T-1- PE2 which was conserved in our experiment. The result of endonuclease Dra I digesting PCR product showed that this gene fragment has the same restriction enzyme site as known CSFV E2 gene. Gene fragments were ligated to pMD18-T vector after purification, the recombinant plasmid pMD18-T-TE2 was transformed into Escherichia coli (E.coli)DH5α,the recombinants were analyzed after they were identified by PCR, subsequently. This suggests that the CSFV TE2 gene has been successfully cloned.
Secondly, gene-specific primers were designed based on the cloned TE2 nucleotide sequences and multi-clone restriction enzyme sites of prokaryotic expressive vector pGEX-4T-l, one gene fragment which encode major antigenic domains of E2(A、B、C、D) about 622bp was amplified from a recombinant plasmid pMD18-T-TE2 by PCR. Then the cDNA fragment which encode major antigenic domains of E2(A、B、C、D) was cloned into pGEX-4T-l,the recombinant plasmid PGEX-4T-1-TE2 was identified by PCR and endonuclease EcoR I +Sal I digestion. The PGEX-4T-1-TE2 was transformed into E.coli Rosetta BL21 DE3 and GST-TE2 fusion protein was induced to express by isopropylthio-β-D-galactoside(IPTG). The MW of the fusion protein was about 47 000 as analyzed by SDS-PAGE.After optimizing prokaryotic expression conditions, we determined the optimum inducement time and concentration of IPTG. The solubility of GST-TE2 fusion protein was identified and the result indicated that expressed GST-TE2 protein existed as inclusion bodies. The recombinant plasmid was induced to express large-scale GST-TE2 fusion protein under the optimal condition, the induced recombinant bacteria was lysed by freeze-thaw and sonication. We obtained the GST-TE2 inclusion body protein, which could be solubilized by sonication after the detergent lauroylsarcosine was added. The concentration of fusion protein GST-TE2 was about 1.2mg/ml by folin-hydroxybenzene.
Finally, using the GST-TE2 fusion protein as antigen, polyclonal antiserum to CSFV E2 was derived from mouse. Results of agar diffusion assay demonstrated that the polyclonal antibody could well react to TE2 protein. ELISA indicated that the immunized rabbits had antibody titers of 1:25600.Western-blotting showed that polyclonal antibody against CSFV GST-TE2 from mouse contain antibody against GST and antibody against GST-TE2 fusion protein.
In conclusion, we have successfully cloned、expressed CSFV TE2 gene, and obtained the polyclonal antibody against CSFV TE2.A11 these provide some experimental materials for the future studies on the immunological function of CSFV E2.
引文
[1]殷震,刘景华.动物病毒学(第二版)[M].北京:科学出版社,1985,517-532
[2]陆承平主编.兽医微生物学[M].北京:中国农业出版社,2001,9,572
[3]白建,曹靖,黄素珍等.猪瘟的研究[M].肉类工业,2005,3:37-40
[4]蔡宝祥主编.家畜传染病学[M].北京:中国农业出版社,1999,147-151
[5]Arjan Stegeman,Armin Elbers,Hans de Smit,et al.The 1997-1998 epidemic of classical swine fever in the Netherlands[J].Veterinary Microbiology,2000,73(2～3):183-196.
[6]SaatkampH W,HorstH S.Economic aspects of the control of classical swine fever[J].Vet Microbiol,2000,73(2-3):221-238.
[7]Dewulf J,Laevens H,Koenen F,et al.An experimental infection with classical swine fever virus in pregnant sows:transmission of the virus,course of the disease,antibody response and effect on gestation[J].J Vet Med B Infect Dis Vet Public Health,2001,48(8):583-591.
[8]王镇,阂光伟,李明义等.猪瘟病毒的形态结构与形态发生[J].微生物学报,2000,40(3):237-242.
[9]HarknessJ W.Classical swine fever and its diagnosis:A current view[J].Vet Rec,1985,16:288-293.
[10]Trawinski A,Trawinska J.Wplyw pH wirulencje wirusu pomoru w miesie swinskim.Med Wet,1949,5:416-417.
[11]Moennig V.Characteristics of the virus.In:Liess,B.(Ed.)Classical swine fever and related in fections[J].Martinus Nijhof,Boston,Dordrecht,Lancaster,1998,pp55-80.
[12]Russell A D,Hugo W B.Chemical disinfectants.In:Linton,AH,Hugo WB,Rus sell AD (Eds),Disinfection in veterinary and farm animal practice[J].Blackwell scientific publications,Oxford,1987,12-42.
[13]杜念兴.猪瘟的感染与免疫[J].畜牧与兽医,1991,23(2):85-88
[14]卫广森.猪瘟研究新进展[J].辽宁畜牧兽医.2004,9:41-46
[15]Meyers G et al.Molecular cloning and nucleotide sequence of the genome of hog cholera virus[J].Virology,1989,171:556-567
[16]王镇,闵光伟,李明义,等.猪瘟病毒的形态结构与形态发生.微生物学报[J].2000,40(3):237-242
[17]王镇,陆宇,周鹏程,等.猪瘟病毒在PK细胞和MPK细胞中繁殖过程中的研究[J].微生物学报.1999,39(3):189-195
[18]王镇,陆宇,丁明孝.猪瘟病毒中国兔化弱毒疫苗株在原代牛睾丸细胞中增殖特性的研究[J].中国病毒学.2000,15(2):170-179
[19]王镇,陆宇,丁明孝.猪瘟病毒弱毒株感染对体外培养细胞增殖的促进作用[J].微生物学报.2000,27(2):79-84
[20]张靖飞.猪瘟病毒致病机制研究进展[J].动物医学进展,2001,22(3):85-86
[21]LeeW C,Wang A S,Chien M S.Virus antigen expression and alterations in peripheral blood Mononuclear cell subpopulations after classical swine fever virus infection.Vet Microbiol,1999,67(1):17-29.
[22]Markowska-Daniel I,Collins R A,Pejsak Z.Evaluation of genetic vaccine against classical swine fever.Vaccine,2001,(17-19):2480-2487.
[23]Summerfield A,Hofmann M A,Mccullough K C,et al.Low density blood granulocytic cells induced during classical swine fever are targets for virus infection[J].Vet Immunol Immunop -athol,1998,63(3):289-301.
[24]Summerfield A,Knotig S M,Mecullough K C.Pathogenesis of granulocytopenia and bone marrow atrophy during classical swine fever involves apoptosis and necrosis of uninfected cells[J].J Virol,1998,72(3):1853-1881
[25]Knoetig S M,Summerfield A,Spagnuolo-Weaver M,et al.Immunopathogenesis of classical swine fever:role of monocytic cells[J].Immunology,1999,97(2),359-366
[26]Rijnbrand R,VanderStraaten T,VanRijn P A,et al.Internal entry of ribosome is directed by the 5' noncoding region of Classical Swine Fever Virus and is dependent on the presence of an RNA pseudoknot upstream of the the intiation codon[J].J.Virol,1997,71(1):451-457.
[27]Meyers G,T Rmenapf,H-J Thiel.Molecular cloning and nucleotide sequence of the genome of Hog Cholera virus[J].Virology,1989,171:555-567
[28]Moonnann R J M,Van Gen H G P,Miedema G K W,et al.Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus[J],virol,1996,2:736-770
[29]Stark R,Rumcnapf T,G Meyers.Genomic localization of Hog Cholera virus glyvoproteins[J].Virology,1990,174:286-289
[30]Kromykh A A,E G Westwy.Completion of kunjin virus RNA sequence and recovery of Infectious RNA transcriped from stably cloned full-length cDNA[J].Virol,1994,68:4580-4591
[31]Ishkawa K,Nagai H,Katayama K,et al.Copararison of the entire deduced amino acid sequence of the attenuated hog cholera vaccine strain GPE~- and the widetype parental strain ALD[J].Arch.Virology,1995,140:1385-1391
[32]Ruggli N,Tratschin J,Mitelholzer C.Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA[J].Virus,1996.6:3478-3487
[33]李红卫等.HCV兔化弱毒株5'端调控序列及P23基因的扩增与克隆[J].中国兽医学报,1998,(15)4:320-323
[34]李红卫等.HCV石门株与兔化弱毒株兔脾组织毒gP55基因的克隆与序列分析[J].病毒学报,1998,(14)3:257-161
[35]韩雪清.中国HCV兔化弱毒株兔脾组织毒部分基因的序列分析[J].中国兽医科技,1998,(28)6:12-16
[36]黄茜华等.HCV石门株NS4B基因的序列测定及分析[J].湖北农学院学报,1999,19:53-55
[37]黄茜华等.HCV石门株NS5A基因的序列测定及同源比较[J].华中农业大学学报,1999,(18)2:154-158
[38]李红卫,刘湘涛,李小兵等.我国猪瘟病毒兔化弱毒株囊膜糖蛋白EO基因的克隆及序列测定[J].中国病毒学,1999,14(2):169-173
[39]Joseph M,Fernandez,James P Hoeffler.Gene Expression Systems.New-york,Academic Press,1999.
[40]Yu XL,Tu CC,Li H W et al.DNA-mediated protein against classical swine fever virus[J].Vaccine,2001,19(11-12):1520.
[41]Greiser-Wilke I,Moennig V,Liess B,Identification of conserved epitopes on a hog cholera virus protein[J].Arch Virol,1990,111:213.
[42]Welland E,Stark R,Haas B et al.Pestivirus glycoprotein which induces neutralizing antibodies forms part of disulfide-linked heterodimer[J].J Virol,1990,64:3563.
[43]Van Rijin P A,Bossers A,Wensvoort G et al.Classical Swine Fever Virus envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge[J].J Gen Virol,1996,77:2737.
[44]Hulst M M,Westra D F,Wensvoort G et al.Glycoprotein El of Hog Cholera Virus expressed in insect cells protects Swine from Hog Cholera[J].J of Virol,1993,67(9):5435.
[45]Mulder W A M,Priem J,Glaznbura K,et al.Virulence and pathogensis of nonvirulent strains of psedorabirs virus expressing envelop glycoprteins El of hog cholera virus[J].Gen virol,1994,75:117-124
[46]Hulst M M,Wensvoort G,et al.Glycoprteins El of hog cholera virus expressed in insect cells protects swine hog cholera[J].virol,1993,67:5434-5442
[47]Vilcek S,Stadeje K T,Takacsova I,et al.Genetic analysis of classical swine fever virus isolates from a small geographic area.DTW Dtsd Tierarztl wochenschr[J].1997,104:9-12
[48]Van Rijn P A,H G P Van Gennip,E J M Moormann,et al.Eptitope mapping of envelope glycoprotein El of hog cholera virus strain Brescia[J].Gen Virol 1993,74:2053-2060
[49]Van Rijn P A,Van Gennip H G P,De meijer E J,et al.A preliminary map of epitopes on enveliope glycoprtein El of hog cholera virus strain brescia[J].Vet Micro,1992,33:221-230
[50]Rumenapf T,Ungher G,Strauss J H,et al.Processing of the envelope glycoproteins of pestivirus [J].Virol,1993,67:3288-3294
[51]Kimman T G,Bianchi A T J,Wensvoort G,et al.Cellular immune response to hog cholera virus(HCV):T cells of immune pigs proliferate in vitro upon stimu- lation with live HCV ,but the El envelope glycoprotein is not a major T-cell antigen[J].J Virol,1993,67:2922-2927
[52]Wensvoort G.Topographical and functional maping epitomes on hog cholera virus with mon-oclone antibody[J].Gen Virus,1989
[53]Van Rijn P A,G K W Miedema,G Wensvoort,et al.Antigenic structure of envelope glycop-rotein El of hog cholera virus[J].Virol,1994,75:3934-3942
[54]Van Rijn P A,A Bossers,G Wensvoort,et aI.Classical swine fever virus envelope glycoprotein E2 containing one structure antigenic unit protects swine from lethal CSFV challenge[J].Gen Virol,1996,77:2737-2745
[55]Min lin,Fang Lin,M Mallery,et al.Deletion of structural glycoprotein E2 of Class-ical swine fever virus srtrain Alfort1187 resolve a linear epitops of monoclonal antibodies WH303 and N-terminal domain essential for binding immunoglob-ulin G antibodies of a pig hyperimmune serum[J].Virol.,2000,11619-11625
[56]王宁,傅烈增,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(1):74-78.
[57]傅烈振,朱燕,王家富等.猪瘟病毒HCLV株E2核苷酸序列与结构分析[J].武汉大学学报(自然科学版),1999,45(4):509-512.
[58]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].中国兽医学报,1998,18(2):112-114.
[59]满朝来,李一经.猪瘟病毒国内分离株(HL-LY)E2基因的克隆及序列分析[J].黑龙江畜牧兽医,2003,2:3-4.
[60]李红卫,余兴龙,涂长春等.以重组mE2蛋白为抗原建立检测猪瘟病毒抗体间接ELISA方法的研究[J].中国预防兽医学报,1999,21(3):220-222.
[61]王宁,张楚瑜,付烈振等.猪瘟病毒石门株E2基因的克隆及在大肠杆菌中的表达[J].武汉大学学报(自然科学版),1998,44(2):259-261.
[62]张永国,刘湘涛,韩雪清.猪瘟病毒E2基因抗原结构域A、B、C、D区在大肠杆菌中的表达[J].畜牧兽医学报,2004,35(2):182-185.
[63]余兴龙,涂长春,徐兴然.猪瘟病毒E2基因的定点突变、在大肠杆菌中的高效表达及表达产物的免疫原性[J].生物工程学报,2003,19(4):439-443.
[64]范学政,王琴,陈振海.猪瘟兔化弱毒E2基因的原核表达及间接ELISA的初步建立[J].中国病毒学,2005,3:253-256.
[65]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,2000,15(3):264-271.
[66]陈创夫,余兴龙,乔军等.IL—2与猪瘟病毒E2基因真核双表达栽体的构建及其免疫增强作用的研究[J].石河子大学学报,2001,5(2):90-94.
[67]Berns A.Live atenuated pseudorabies virus expressing envelope glyloprotein El of hog cholera virus protects swine against both pseudorabies and hog cholera virus[J].J Virol,1991,65:2761-2765.
[68]Hulst M M,Westra D F,Wensvoort Get al.Glycoprotein El of Hog Cholera Virus expressed in insect cells protects Swine from Hog Cholera[J].J of Virol,1993,67(9):5435.
[69]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
[70]Mulder W,Priern J,Glazenbura K,et al.Virulence and pathogensis of nonvirulent and strains of pseudorabies virus expressing envelope glycoproteim El Hog cholera virus[J].J Gen Virol.1994,75:117-124.
[71]Yu X L,Tu C C,Li H W,et al.DNA-mediated protection against classical swine fever virus [J].Vaccine,2001,19:1520-1525.
[72]Hammond J M,Jansen E S,Morrissy C J,et al.A prime-boost vaccination strategy using naked DNA followed by recombinant porcine adenovirus protects pigs from classical swine fever[J].Vet Microbiol,2001,80(2):101-119.
[73]刘思国.猪瘟病毒E2蛋白抗原表位研究博士后研究报告.2001,长春.
[74]赵志军.猪瘟病毒E2基因克隆、序列分析及最佳猪瘟免疫程序的制定:[硕士学位论文].石家庄:河北农业大学,2003
[75]Becher P,Avalos Ramirez R,Orlich M et al.Genetic and antigenic characterization of novel pestivirus genotypes:implications for classification[J].Virology,2003,311(1):96-104.
[76]Van Riin P A,Miedema G K W,Wensveort G,et al.Antigenic stucture of envelope glycoprotein El of hog cholera virus[J].J Virol,1994,68:3934-3942.
[77]Van RijnP A,de Meijer E J,van Gennip H G P,et al.Epitope mapping of envelope glycoprotein EI of hog cholen virus strain Brescia[J].J Gen Virol,1993,74:2053-2060.
[78]C.W.迪芬巴赫,G.S德维克斯勒 著,黄培堂,愈炜源,陈添源等 译.PCR技术实验指南[M].北京:科学出版社,2000.
[79]J 萨姆布鲁克(美)著,金冬雁,黎孟枫等 译.分子克隆实验指南(第二版)[M].北京:科学出版社.1992
[80]J.萨母布鲁克(美),D.W拉塞尔(美)著,黄培堂 译.分子克隆实验指南(第三版)[M].北京:科学出版社.2002.1217-1259
[81]Garroll,Laughon.In DNA cloning:a practical approach.IRL.A.S.C,1987,3:89-111
[82]Frangioni JV,Neel BG.Solubilization and purification of enzymatically active glutathione s-transferase.Anal Biochem,1991,192:262-267.
[83]Melania E.Mercado-Pimentel,Nicole C.et al.Affinity purification of GST fusion proteins for immunohistochemical studies of gene expression.Science,2002,26:260-265.
[84]秦卫松.穿孔素氨基端肽段的原核表达、抗体制备和信使核糖核酸的定量检测:[硕士学位论文].上海:第二军医大学,2001.
[85]J.萨姆布鲁克,E.F弗里奇,T.曼尼阿蒂斯.分子克隆实验指南(第二版)[M].北京:科学出版社,1996:888-897.
[86]姜泊等主编.分子生物学常用实验方法(第二版)[M].人民军医出版社.1996年2月.
[87]卢圣栋主编.现代分子生物学实验技术(第二版)[M].北京:中国协和医科大学出版社,1999,378-379
[88]Busuttil B E,Tumey K L,Frauman A G.Protein Expression and Purification,2001,23(3):369-373
[89]Leandro P,Lechner M C,,Almerda H,et al.Molecular Genetics and Metabolism,2001,73(2):173-178
[90]Knrosawa Y.Development of a prokarytic expression vector that exploits dicitronic gene organizationnrn[J].Gene,1992,118:87-91
[91]Guan K L,Dixon J E,Eukaryotic proteins expressed in Escherichia coli:an improved thrombin cleavage and purification procedure of fusion proteins with gluthione S- transferase[J].Anal Biochem,1991,192:262-267
[92]Studier FW,Rosenberg A H,Dunn J J,et al.Use of T7 RNA polymerase to direct expression of cloned genes[J].Methods Enzymol.1990,85:60-89
[93]Dubendorff,Studier.Creation of a T7 autogene.Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter[J].J Mol Biol.1991,219(1):61-68
[94]张锐,孙美榕,欧阳红生等.真核基因在pET系统中表达出现的问题与拟解决的方案[J].生物技术,2004,14(2):62-63.
[95]Grosjean H,Fiers W.Gene,1982,18:199-209
[96]Sung W L,Zahab D M,Barbier J R,et al.J Biol Chem,1991,266(5):2831-2835
[97]Srinivasan G,James C M,Krzycki J A.Science,2002,296(5572):1459-1462
[98]吴乃虎.基因工程原理[M].北京:科学出版社,2001,117-119.
[99]余贺,谢少文,杨贵贞.临床免疫技术[M].上海科学技术出版社,1982:437-440.
[2]陆承平主编.兽医微生物学[M].北京:中国农业出版社,2001,9,572
[3]白建,曹靖,黄素珍等.猪瘟的研究[M].肉类工业,2005,3:37-40
[4]蔡宝祥主编.家畜传染病学[M].北京:中国农业出版社,1999,147-151
[5]Arjan Stegeman,Armin Elbers,Hans de Smit,et al.The 1997-1998 epidemic of classical swine fever in the Netherlands[J].Veterinary Microbiology,2000,73(2～3):183-196.
[6]SaatkampH W,HorstH S.Economic aspects of the control of classical swine fever[J].Vet Microbiol,2000,73(2-3):221-238.
[7]Dewulf J,Laevens H,Koenen F,et al.An experimental infection with classical swine fever virus in pregnant sows:transmission of the virus,course of the disease,antibody response and effect on gestation[J].J Vet Med B Infect Dis Vet Public Health,2001,48(8):583-591.
[8]王镇,阂光伟,李明义等.猪瘟病毒的形态结构与形态发生[J].微生物学报,2000,40(3):237-242.
[9]HarknessJ W.Classical swine fever and its diagnosis:A current view[J].Vet Rec,1985,16:288-293.
[10]Trawinski A,Trawinska J.Wplyw pH wirulencje wirusu pomoru w miesie swinskim.Med Wet,1949,5:416-417.
[11]Moennig V.Characteristics of the virus.In:Liess,B.(Ed.)Classical swine fever and related in fections[J].Martinus Nijhof,Boston,Dordrecht,Lancaster,1998,pp55-80.
[12]Russell A D,Hugo W B.Chemical disinfectants.In:Linton,AH,Hugo WB,Rus sell AD (Eds),Disinfection in veterinary and farm animal practice[J].Blackwell scientific publications,Oxford,1987,12-42.
[13]杜念兴.猪瘟的感染与免疫[J].畜牧与兽医,1991,23(2):85-88
[14]卫广森.猪瘟研究新进展[J].辽宁畜牧兽医.2004,9:41-46
[15]Meyers G et al.Molecular cloning and nucleotide sequence of the genome of hog cholera virus[J].Virology,1989,171:556-567
[16]王镇,闵光伟,李明义,等.猪瘟病毒的形态结构与形态发生.微生物学报[J].2000,40(3):237-242
[17]王镇,陆宇,周鹏程,等.猪瘟病毒在PK细胞和MPK细胞中繁殖过程中的研究[J].微生物学报.1999,39(3):189-195
[18]王镇,陆宇,丁明孝.猪瘟病毒中国兔化弱毒疫苗株在原代牛睾丸细胞中增殖特性的研究[J].中国病毒学.2000,15(2):170-179
[19]王镇,陆宇,丁明孝.猪瘟病毒弱毒株感染对体外培养细胞增殖的促进作用[J].微生物学报.2000,27(2):79-84
[20]张靖飞.猪瘟病毒致病机制研究进展[J].动物医学进展,2001,22(3):85-86
[21]LeeW C,Wang A S,Chien M S.Virus antigen expression and alterations in peripheral blood Mononuclear cell subpopulations after classical swine fever virus infection.Vet Microbiol,1999,67(1):17-29.
[22]Markowska-Daniel I,Collins R A,Pejsak Z.Evaluation of genetic vaccine against classical swine fever.Vaccine,2001,(17-19):2480-2487.
[23]Summerfield A,Hofmann M A,Mccullough K C,et al.Low density blood granulocytic cells induced during classical swine fever are targets for virus infection[J].Vet Immunol Immunop -athol,1998,63(3):289-301.
[24]Summerfield A,Knotig S M,Mecullough K C.Pathogenesis of granulocytopenia and bone marrow atrophy during classical swine fever involves apoptosis and necrosis of uninfected cells[J].J Virol,1998,72(3):1853-1881
[25]Knoetig S M,Summerfield A,Spagnuolo-Weaver M,et al.Immunopathogenesis of classical swine fever:role of monocytic cells[J].Immunology,1999,97(2),359-366
[26]Rijnbrand R,VanderStraaten T,VanRijn P A,et al.Internal entry of ribosome is directed by the 5' noncoding region of Classical Swine Fever Virus and is dependent on the presence of an RNA pseudoknot upstream of the the intiation codon[J].J.Virol,1997,71(1):451-457.
[27]Meyers G,T Rmenapf,H-J Thiel.Molecular cloning and nucleotide sequence of the genome of Hog Cholera virus[J].Virology,1989,171:555-567
[28]Moonnann R J M,Van Gen H G P,Miedema G K W,et al.Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus[J],virol,1996,2:736-770
[29]Stark R,Rumcnapf T,G Meyers.Genomic localization of Hog Cholera virus glyvoproteins[J].Virology,1990,174:286-289
[30]Kromykh A A,E G Westwy.Completion of kunjin virus RNA sequence and recovery of Infectious RNA transcriped from stably cloned full-length cDNA[J].Virol,1994,68:4580-4591
[31]Ishkawa K,Nagai H,Katayama K,et al.Copararison of the entire deduced amino acid sequence of the attenuated hog cholera vaccine strain GPE~- and the widetype parental strain ALD[J].Arch.Virology,1995,140:1385-1391
[32]Ruggli N,Tratschin J,Mitelholzer C.Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA[J].Virus,1996.6:3478-3487
[33]李红卫等.HCV兔化弱毒株5'端调控序列及P23基因的扩增与克隆[J].中国兽医学报,1998,(15)4:320-323
[34]李红卫等.HCV石门株与兔化弱毒株兔脾组织毒gP55基因的克隆与序列分析[J].病毒学报,1998,(14)3:257-161
[35]韩雪清.中国HCV兔化弱毒株兔脾组织毒部分基因的序列分析[J].中国兽医科技,1998,(28)6:12-16
[36]黄茜华等.HCV石门株NS4B基因的序列测定及分析[J].湖北农学院学报,1999,19:53-55
[37]黄茜华等.HCV石门株NS5A基因的序列测定及同源比较[J].华中农业大学学报,1999,(18)2:154-158
[38]李红卫,刘湘涛,李小兵等.我国猪瘟病毒兔化弱毒株囊膜糖蛋白EO基因的克隆及序列测定[J].中国病毒学,1999,14(2):169-173
[39]Joseph M,Fernandez,James P Hoeffler.Gene Expression Systems.New-york,Academic Press,1999.
[40]Yu XL,Tu CC,Li H W et al.DNA-mediated protein against classical swine fever virus[J].Vaccine,2001,19(11-12):1520.
[41]Greiser-Wilke I,Moennig V,Liess B,Identification of conserved epitopes on a hog cholera virus protein[J].Arch Virol,1990,111:213.
[42]Welland E,Stark R,Haas B et al.Pestivirus glycoprotein which induces neutralizing antibodies forms part of disulfide-linked heterodimer[J].J Virol,1990,64:3563.
[43]Van Rijin P A,Bossers A,Wensvoort G et al.Classical Swine Fever Virus envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge[J].J Gen Virol,1996,77:2737.
[44]Hulst M M,Westra D F,Wensvoort G et al.Glycoprotein El of Hog Cholera Virus expressed in insect cells protects Swine from Hog Cholera[J].J of Virol,1993,67(9):5435.
[45]Mulder W A M,Priem J,Glaznbura K,et al.Virulence and pathogensis of nonvirulent strains of psedorabirs virus expressing envelop glycoprteins El of hog cholera virus[J].Gen virol,1994,75:117-124
[46]Hulst M M,Wensvoort G,et al.Glycoprteins El of hog cholera virus expressed in insect cells protects swine hog cholera[J].virol,1993,67:5434-5442
[47]Vilcek S,Stadeje K T,Takacsova I,et al.Genetic analysis of classical swine fever virus isolates from a small geographic area.DTW Dtsd Tierarztl wochenschr[J].1997,104:9-12
[48]Van Rijn P A,H G P Van Gennip,E J M Moormann,et al.Eptitope mapping of envelope glycoprotein El of hog cholera virus strain Brescia[J].Gen Virol 1993,74:2053-2060
[49]Van Rijn P A,Van Gennip H G P,De meijer E J,et al.A preliminary map of epitopes on enveliope glycoprtein El of hog cholera virus strain brescia[J].Vet Micro,1992,33:221-230
[50]Rumenapf T,Ungher G,Strauss J H,et al.Processing of the envelope glycoproteins of pestivirus [J].Virol,1993,67:3288-3294
[51]Kimman T G,Bianchi A T J,Wensvoort G,et al.Cellular immune response to hog cholera virus(HCV):T cells of immune pigs proliferate in vitro upon stimu- lation with live HCV ,but the El envelope glycoprotein is not a major T-cell antigen[J].J Virol,1993,67:2922-2927
[52]Wensvoort G.Topographical and functional maping epitomes on hog cholera virus with mon-oclone antibody[J].Gen Virus,1989
[53]Van Rijn P A,G K W Miedema,G Wensvoort,et al.Antigenic structure of envelope glycop-rotein El of hog cholera virus[J].Virol,1994,75:3934-3942
[54]Van Rijn P A,A Bossers,G Wensvoort,et aI.Classical swine fever virus envelope glycoprotein E2 containing one structure antigenic unit protects swine from lethal CSFV challenge[J].Gen Virol,1996,77:2737-2745
[55]Min lin,Fang Lin,M Mallery,et al.Deletion of structural glycoprotein E2 of Class-ical swine fever virus srtrain Alfort1187 resolve a linear epitops of monoclonal antibodies WH303 and N-terminal domain essential for binding immunoglob-ulin G antibodies of a pig hyperimmune serum[J].Virol.,2000,11619-11625
[56]王宁,傅烈增,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(1):74-78.
[57]傅烈振,朱燕,王家富等.猪瘟病毒HCLV株E2核苷酸序列与结构分析[J].武汉大学学报(自然科学版),1999,45(4):509-512.
[58]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].中国兽医学报,1998,18(2):112-114.
[59]满朝来,李一经.猪瘟病毒国内分离株(HL-LY)E2基因的克隆及序列分析[J].黑龙江畜牧兽医,2003,2:3-4.
[60]李红卫,余兴龙,涂长春等.以重组mE2蛋白为抗原建立检测猪瘟病毒抗体间接ELISA方法的研究[J].中国预防兽医学报,1999,21(3):220-222.
[61]王宁,张楚瑜,付烈振等.猪瘟病毒石门株E2基因的克隆及在大肠杆菌中的表达[J].武汉大学学报(自然科学版),1998,44(2):259-261.
[62]张永国,刘湘涛,韩雪清.猪瘟病毒E2基因抗原结构域A、B、C、D区在大肠杆菌中的表达[J].畜牧兽医学报,2004,35(2):182-185.
[63]余兴龙,涂长春,徐兴然.猪瘟病毒E2基因的定点突变、在大肠杆菌中的高效表达及表达产物的免疫原性[J].生物工程学报,2003,19(4):439-443.
[64]范学政,王琴,陈振海.猪瘟兔化弱毒E2基因的原核表达及间接ELISA的初步建立[J].中国病毒学,2005,3:253-256.
[65]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,2000,15(3):264-271.
[66]陈创夫,余兴龙,乔军等.IL—2与猪瘟病毒E2基因真核双表达栽体的构建及其免疫增强作用的研究[J].石河子大学学报,2001,5(2):90-94.
[67]Berns A.Live atenuated pseudorabies virus expressing envelope glyloprotein El of hog cholera virus protects swine against both pseudorabies and hog cholera virus[J].J Virol,1991,65:2761-2765.
[68]Hulst M M,Westra D F,Wensvoort Get al.Glycoprotein El of Hog Cholera Virus expressed in insect cells protects Swine from Hog Cholera[J].J of Virol,1993,67(9):5435.
[69]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
[70]Mulder W,Priern J,Glazenbura K,et al.Virulence and pathogensis of nonvirulent and strains of pseudorabies virus expressing envelope glycoproteim El Hog cholera virus[J].J Gen Virol.1994,75:117-124.
[71]Yu X L,Tu C C,Li H W,et al.DNA-mediated protection against classical swine fever virus [J].Vaccine,2001,19:1520-1525.
[72]Hammond J M,Jansen E S,Morrissy C J,et al.A prime-boost vaccination strategy using naked DNA followed by recombinant porcine adenovirus protects pigs from classical swine fever[J].Vet Microbiol,2001,80(2):101-119.
[73]刘思国.猪瘟病毒E2蛋白抗原表位研究博士后研究报告.2001,长春.
[74]赵志军.猪瘟病毒E2基因克隆、序列分析及最佳猪瘟免疫程序的制定:[硕士学位论文].石家庄:河北农业大学,2003
[75]Becher P,Avalos Ramirez R,Orlich M et al.Genetic and antigenic characterization of novel pestivirus genotypes:implications for classification[J].Virology,2003,311(1):96-104.
[76]Van Riin P A,Miedema G K W,Wensveort G,et al.Antigenic stucture of envelope glycoprotein El of hog cholera virus[J].J Virol,1994,68:3934-3942.
[77]Van RijnP A,de Meijer E J,van Gennip H G P,et al.Epitope mapping of envelope glycoprotein EI of hog cholen virus strain Brescia[J].J Gen Virol,1993,74:2053-2060.
[78]C.W.迪芬巴赫,G.S德维克斯勒 著,黄培堂,愈炜源,陈添源等 译.PCR技术实验指南[M].北京:科学出版社,2000.
[79]J 萨姆布鲁克(美)著,金冬雁,黎孟枫等 译.分子克隆实验指南(第二版)[M].北京:科学出版社.1992
[80]J.萨母布鲁克(美),D.W拉塞尔(美)著,黄培堂 译.分子克隆实验指南(第三版)[M].北京:科学出版社.2002.1217-1259
[81]Garroll,Laughon.In DNA cloning:a practical approach.IRL.A.S.C,1987,3:89-111
[82]Frangioni JV,Neel BG.Solubilization and purification of enzymatically active glutathione s-transferase.Anal Biochem,1991,192:262-267.
[83]Melania E.Mercado-Pimentel,Nicole C.et al.Affinity purification of GST fusion proteins for immunohistochemical studies of gene expression.Science,2002,26:260-265.
[84]秦卫松.穿孔素氨基端肽段的原核表达、抗体制备和信使核糖核酸的定量检测:[硕士学位论文].上海:第二军医大学,2001.
[85]J.萨姆布鲁克,E.F弗里奇,T.曼尼阿蒂斯.分子克隆实验指南(第二版)[M].北京:科学出版社,1996:888-897.
[86]姜泊等主编.分子生物学常用实验方法(第二版)[M].人民军医出版社.1996年2月.
[87]卢圣栋主编.现代分子生物学实验技术(第二版)[M].北京:中国协和医科大学出版社,1999,378-379
[88]Busuttil B E,Tumey K L,Frauman A G.Protein Expression and Purification,2001,23(3):369-373
[89]Leandro P,Lechner M C,,Almerda H,et al.Molecular Genetics and Metabolism,2001,73(2):173-178
[90]Knrosawa Y.Development of a prokarytic expression vector that exploits dicitronic gene organizationnrn[J].Gene,1992,118:87-91
[91]Guan K L,Dixon J E,Eukaryotic proteins expressed in Escherichia coli:an improved thrombin cleavage and purification procedure of fusion proteins with gluthione S- transferase[J].Anal Biochem,1991,192:262-267
[92]Studier FW,Rosenberg A H,Dunn J J,et al.Use of T7 RNA polymerase to direct expression of cloned genes[J].Methods Enzymol.1990,85:60-89
[93]Dubendorff,Studier.Creation of a T7 autogene.Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter[J].J Mol Biol.1991,219(1):61-68
[94]张锐,孙美榕,欧阳红生等.真核基因在pET系统中表达出现的问题与拟解决的方案[J].生物技术,2004,14(2):62-63.
[95]Grosjean H,Fiers W.Gene,1982,18:199-209
[96]Sung W L,Zahab D M,Barbier J R,et al.J Biol Chem,1991,266(5):2831-2835
[97]Srinivasan G,James C M,Krzycki J A.Science,2002,296(5572):1459-1462
[98]吴乃虎.基因工程原理[M].北京:科学出版社,2001,117-119.
[99]余贺,谢少文,杨贵贞.临床免疫技术[M].上海科学技术出版社,1982:437-440.