猪瘟和口蹄疫病毒抗原基因在植物内的重组与表达
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摘要
口蹄疫和猪瘟都是当今世界上严重危害家畜的烈性传染病,发病率高,流行
快,危害严重。目前,疫苗接种是预防口蹄疫和猪瘟病毒发病的主要手段。传统
的灭活疫苗和弱毒疫苗在防治和消除这两种疾病的大规模流行中发挥了重大作
用。但随着传代次数的增加和对不同细胞的适应性增殖,疫苗毒株可能发生抗原
漂移,加之数十年来病毒在大规模免疫接种压力下出现抗原变异,现有的传统
疫苗已不能适应新形势的需要。而且由于其潜在的危险性,使其应用受到很大限
制。随着分子生物学和分子免疫学的深入发展,迫切需要研制新型的基因工程疫
苗。
转基因植物作为生产疫苗的生物反应器,具有表达量高,成本低,容易生产
等优点。另外,植物表达的抗原蛋白不需要纯化,可以作为口服疫苗直接食用。
如:番茄,大豆,牧草,烟草等。VPl和E2分别是口蹄疫病毒和猪瘟病毒的核心
抗原决定簇,能够诱导机体产生保护性抗体反应。研究表明,VPl和E2已经在多
种表达系统内得到了表达。如:烟草和苜蓿表达的VPl抗原蛋白喂饲小鼠后,能
够诱导特异性抗体免疫反应。为此,本文中将口蹄疫病毒抗原基因VPl和黏膜免
疫佐剂霍乱毒素B亚基(CTB)融合基因在马铃薯内重组和表达;同时,利用植物
叶绿体基因组高效表达的特点,把猪瘟病毒抗原基因E2重组并转化进烟草和衣藻
叶绿体基因组中,使其高效表达。为利用植物生物反应器研制口蹄疫和猪瘟基因
工程疫苗提供理论和实践依据。
论文的第二部分研究了将口蹄疫病毒抗原基因VPl和霍乱毒素B亚基融合基因
通过农杆菌转染的方法转化到马铃薯基因组中,进行重组和表达。通过抗性筛选,
得到了具有卡那霉素抗性的转化植株。对马铃薯基因组DNA的PCR扩增、SotJthern
blot、Western blot和ELISA分析表明,外源基因已经整合进马铃薯基因组,并
得到了表达。
论文的第三部分主要研究了烟草叶绿体表达载体PTRE2的构建,并通过基因枪
法转化烟草叶绿体基因组,通过壮观霉素抗性筛选,获得了一株转化植株苗。经
过两轮抗生素筛选后,分别用CSFV的E2基因及烟草叶绿体同源片段的引物对抗性
植株的叶绿体DNA进行PCR扩增和酶切Southern blot分析,结果表明目的基因已
Both classical swine fever (CSF) and Foot- and- mouth disease (FMD) are highly contagious disease of livestock, which are classified by the Office International des Epizooties (OIE) as a list A disease. Classical swine fever virus (CSFV) and Foot-and-mouth disease virus (FMDV) are the causative agent of classical swine fever (CSF) and Foot- and- mouth disease (FMD) respectively. At present, Vaccine is effective method to prevent the disease. Conventional vaccines are based on the utilization of inactivated and attenuated virus which have considerable risk although they have proved to be effective for the prevention and controlling of the disease. The most widely used conventional vaccination of classical swine fever is The Chinese strain which was safe and effective but interferes with serodiagnosis. So it is necessary to develop safe and effective alternative vaccines.The structural protein VP1 of foot-and-mouth disease virus, which has been shown to contain critical epitopes responsible for the induction of neutralizing antibodies, has been expressed in different expression systems and plants. Production of complete or partial VP1 in a diversity of expression systems has been performed in the search for an effective and inexpensive alternative which would be highly immunogenesis. In this study, we developed a expression vector pBI121CTBVP1 by fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene with a flexible linker tetrapeptide (GPGP) at the c-terminal of CTB, and then cloned into intermediate vector pBI121-patatin containing specific-tuber patatin promoter of potato. Furthermore, we fused the ER targeting signal (SEKDEL) encoding sequence at the c-terminal of VP1 in order to improve expression of fused gene and distribution. The transgenic potatos were obtained by Agrobacterium-mediated transformation and selection on kanamycin resistance agent. The transgenic plantlets were identified by PCR, Southern-blot and the production of fused protein was confirmed and quantified by Western-blot and ELISA assays. The results demonstrated that the fused genes were expressed stablely under control of specific-tuber patatin promoter up to 0.1%-0.13% though it was fant in leaves, and the
transgenic potato could be as oral vaccine candidate defense of foot-and-mouth disease.The E2 protein of CSFV contains major antigenic determinants that is conserved and involved in neutralization by antibodies. The expression of E2 protein in insect and in E. coli has been reported. Animal immunization and the challenge against CSFV experiments have been conducted using the recombinant E2 protein. Here, we studied the expression of CSFV E2 gene of shimen strain in tobacco and C. reinhardti chloroplast by biolistic bombardment transformation. The transformants were identified by PCR, Southern blotting, Western blotting after selection on resistant medium. ELISA quantification assay showed that the expressed E2 protein accumulated up to 1.0-2% and 2% of the total soluble protein in tobacco and C. reinhardti chloroplast respectively. In order to confirm the neutralizing activity of the protected antibodies induced by E2 antigen, neutralization assay were carried out on mices. The results of the study showed that the protein E2 expressed in tobacco and C. reinhardti chloroplast could elicit animal bodies to produce antibodies. This indicated that the expressed E2 proteins have a certain of immunogenicity. Our finding provides a new way to develop plant-drived vaccine against CSFV.
快,危害严重。目前,疫苗接种是预防口蹄疫和猪瘟病毒发病的主要手段。传统
的灭活疫苗和弱毒疫苗在防治和消除这两种疾病的大规模流行中发挥了重大作
用。但随着传代次数的增加和对不同细胞的适应性增殖,疫苗毒株可能发生抗原
漂移,加之数十年来病毒在大规模免疫接种压力下出现抗原变异,现有的传统
疫苗已不能适应新形势的需要。而且由于其潜在的危险性,使其应用受到很大限
制。随着分子生物学和分子免疫学的深入发展,迫切需要研制新型的基因工程疫
苗。
转基因植物作为生产疫苗的生物反应器,具有表达量高,成本低,容易生产
等优点。另外,植物表达的抗原蛋白不需要纯化,可以作为口服疫苗直接食用。
如:番茄,大豆,牧草,烟草等。VPl和E2分别是口蹄疫病毒和猪瘟病毒的核心
抗原决定簇,能够诱导机体产生保护性抗体反应。研究表明,VPl和E2已经在多
种表达系统内得到了表达。如:烟草和苜蓿表达的VPl抗原蛋白喂饲小鼠后,能
够诱导特异性抗体免疫反应。为此,本文中将口蹄疫病毒抗原基因VPl和黏膜免
疫佐剂霍乱毒素B亚基(CTB)融合基因在马铃薯内重组和表达;同时,利用植物
叶绿体基因组高效表达的特点,把猪瘟病毒抗原基因E2重组并转化进烟草和衣藻
叶绿体基因组中,使其高效表达。为利用植物生物反应器研制口蹄疫和猪瘟基因
工程疫苗提供理论和实践依据。
论文的第二部分研究了将口蹄疫病毒抗原基因VPl和霍乱毒素B亚基融合基因
通过农杆菌转染的方法转化到马铃薯基因组中,进行重组和表达。通过抗性筛选,
得到了具有卡那霉素抗性的转化植株。对马铃薯基因组DNA的PCR扩增、SotJthern
blot、Western blot和ELISA分析表明,外源基因已经整合进马铃薯基因组,并
得到了表达。
论文的第三部分主要研究了烟草叶绿体表达载体PTRE2的构建,并通过基因枪
法转化烟草叶绿体基因组,通过壮观霉素抗性筛选,获得了一株转化植株苗。经
过两轮抗生素筛选后,分别用CSFV的E2基因及烟草叶绿体同源片段的引物对抗性
植株的叶绿体DNA进行PCR扩增和酶切Southern blot分析,结果表明目的基因已
Both classical swine fever (CSF) and Foot- and- mouth disease (FMD) are highly contagious disease of livestock, which are classified by the Office International des Epizooties (OIE) as a list A disease. Classical swine fever virus (CSFV) and Foot-and-mouth disease virus (FMDV) are the causative agent of classical swine fever (CSF) and Foot- and- mouth disease (FMD) respectively. At present, Vaccine is effective method to prevent the disease. Conventional vaccines are based on the utilization of inactivated and attenuated virus which have considerable risk although they have proved to be effective for the prevention and controlling of the disease. The most widely used conventional vaccination of classical swine fever is The Chinese strain which was safe and effective but interferes with serodiagnosis. So it is necessary to develop safe and effective alternative vaccines.The structural protein VP1 of foot-and-mouth disease virus, which has been shown to contain critical epitopes responsible for the induction of neutralizing antibodies, has been expressed in different expression systems and plants. Production of complete or partial VP1 in a diversity of expression systems has been performed in the search for an effective and inexpensive alternative which would be highly immunogenesis. In this study, we developed a expression vector pBI121CTBVP1 by fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene with a flexible linker tetrapeptide (GPGP) at the c-terminal of CTB, and then cloned into intermediate vector pBI121-patatin containing specific-tuber patatin promoter of potato. Furthermore, we fused the ER targeting signal (SEKDEL) encoding sequence at the c-terminal of VP1 in order to improve expression of fused gene and distribution. The transgenic potatos were obtained by Agrobacterium-mediated transformation and selection on kanamycin resistance agent. The transgenic plantlets were identified by PCR, Southern-blot and the production of fused protein was confirmed and quantified by Western-blot and ELISA assays. The results demonstrated that the fused genes were expressed stablely under control of specific-tuber patatin promoter up to 0.1%-0.13% though it was fant in leaves, and the
transgenic potato could be as oral vaccine candidate defense of foot-and-mouth disease.The E2 protein of CSFV contains major antigenic determinants that is conserved and involved in neutralization by antibodies. The expression of E2 protein in insect and in E. coli has been reported. Animal immunization and the challenge against CSFV experiments have been conducted using the recombinant E2 protein. Here, we studied the expression of CSFV E2 gene of shimen strain in tobacco and C. reinhardti chloroplast by biolistic bombardment transformation. The transformants were identified by PCR, Southern blotting, Western blotting after selection on resistant medium. ELISA quantification assay showed that the expressed E2 protein accumulated up to 1.0-2% and 2% of the total soluble protein in tobacco and C. reinhardti chloroplast respectively. In order to confirm the neutralizing activity of the protected antibodies induced by E2 antigen, neutralization assay were carried out on mices. The results of the study showed that the protein E2 expressed in tobacco and C. reinhardti chloroplast could elicit animal bodies to produce antibodies. This indicated that the expressed E2 proteins have a certain of immunogenicity. Our finding provides a new way to develop plant-drived vaccine against CSFV.
引文
范国昌,苏宁,张中林等.衣藻叶绿体表达体系的建立 科学通报,1999,44(12):1301-1306.
胡建和,陈永耀,王自良,谢庆阁.新型猪瘟疫苗的研究和应用前景.中国兽医科技.2003,33:36-39.
胡建和,张彦明,谢庆阁.猪瘟病毒的分子生物学研究进展.动物医学进展.2002,23:15-18.
J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南.第三版.北京:科学出版社,2002.27
蒋文俊,李华春,李乐,信爱国,廖德芳 口蹄疫疫苗研究进展 畜牧与兽医,2004,36:42-43.
刘晓松.家畜口蹄疫研究概况及防制措施.内蒙古畜牧科学,2000,21(1):46—48.
刘思国.猪瘟病毒 E2.蛋白抗原表位的研究博士后研究报告.2001,长春.
孔桂美.应用植物表达系统生产疫苗的研究概况.生物技术通.2002,13:226-227.
任志强,刘惠民,李润植,杨慧珍.转基因植物作为生物反应器在疫苗生产中的应用.中国生物工程杂志,2003,23:31-35.
苏宁,沈桂芳.植物基因工程新途径:叶绿体转化 生物技术通报,2001,4:9-13.
杨永钦,李乐.口蹄疫灭活二联苗免疫研究.中国畜禽传染病,1995,82(3):29—30.
尹德华,韩福祥.家畜口蹄疫及其防制.北京:中国农业出版社,1994.
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.中国病毒学,2000,15(3):264～271.
赵启祖,谢庆阁.家畜口蹄疫疫苗简介.中国兽医技术,2000,30(6):43—44.
赵凯,邹仕平,张震宇,等.抗O型口蹄疫病毒DNA疫苗的建立.中国免疫学杂志,2002,18(2):89—92.
赵耘,宁宜宝.猪瘟疫苗研究进展.动物医学进展.2004,25(1):39-42.
周鹏程,陆宇,陈建国.猪瘟病毒E2(gp55)基因的克隆表达及其DNA疫苗的初 步研究.微生物学报,2000,40(3):223～251.
Andrew ME, Morrissy CJ, Lenghaus C, Oke PG, Sproat K.W., Hodgson ALM, Johnson M. A and Coupa BEH.Protection of pigs against classical swine fever with DNA-delivered gp55. Vaccine, 2000, 8: 1932-1938.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 2: 248-54.
Bouma A, De Smit A.J, De Kluijver E.P, Terpstra C and Moormann R. J. Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus. Vet. Microbiol. 1999, 66(2): 101-114.
Barteling SJ, Vreeswijk J. Development in foot-and-mouth disease vaccines. Vaccine, 1992,75-88.
Bittle JL, Houghton PA, Alexancler H, et al. Protection against foot-and-mouth disease by immunization with a Chemically Synthesized peptide predicted from the viral nucleotide sequence. Nature, 1982, 298:30-33.
Brown F. Vaccination against foot and mouth disease virus. Vaccine, 1992, 10:1022-1026.
Brown F. New approaches to vaccine against foot-and-mouth disease. Vaccine, 1988,6:180-183.
Blowers AD.Transcriptional analysis of endogenous and foreign genes in chloroplast transformants of Chlamydomonas. Plant Cell, 1990, 2:1059-1070.
Boynton JE, Gillham NW, Harris EH, et al. Chloroplast transformation inChlamydomonas with high velocity microprojectiles. Science, 1988, 240: 1534-1538.
Carrillo C, Wigdorovitz A, Oliveros JC, et al. Protective immune response tofoot-and-mouth disease virus with VP1 expressed in transgenic plants. J Virol. 1998,72:1688-1690.
Carrillo C, Wigdorovitz A, Trono K, et al. Induction of a virus-specific antibody response to foot and mouth disease virus using the structural protein VP1 expressed in transgenic potato plants. Viral Immunol, 2001,14(1): 49-57
Curtiss RI, Cardineau CA. Oral immunization by transgenic plants. World Patent Application ,1990, WO 90/02484.
Daniell, H. New tools for chloroplast genetic engineering. Nal Biotechnol. 1999,17:855-856.
Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: production of antibodies biopharmaceuticals and edible vaccine in plants. Trends in plant Science. 2001,6:219-226.
Daniell H, Muthukumar B, Lee SB. Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection. Curr Genet. 2001, 39:109-116.
De Cosa B, Moar W, Lee SB, Miller M, Daniell H. Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol. 2001,19:71-74.
Dus Santos MJ, Wigdorovitz A, Trono K, et al. A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants.Vaccine, 2002,20(7-8): 1141-7.
Davies G. Foot and mouth disease. Res Vet Sc, 2002,3(3): 195-9.
De Smit A.J, Bouma A, De Kluijver E.P, Terpstra C and Moormann R.J. Duration ofn the protection of an E2 subunit marker vaccine against classical swine fever after a single vaccination. Vet. Microbiol. 2001, 8 : 307-317.
Depner K.R, Bouma A, Koenen F, Klinkenberg D, Lange E, De Smit J.A and Vanderhallen H. Classical swine fever (CSF) marker vaccine. Trial II. Challenge study in pregnant sows. Vet. Microbiol. 2001, 83: 107-120.
Dong XN, Wei K, Liu ZQ, Chen YH. Candidate peptide vaccine induced protection against classical swine fever virus. Vaccine. 2002, (3-4): 167-73.
Edwards S, Fukusho A and Lefevre P.C. Classical swine fever: the global situation. Vet. Microbiol 2000, 3(2-3): 103-119.
Echtold, N., J. Ellis, and G. Pelletier. Agrobacterium mediated gene transfer by infiltration of adult Arabidpsis thaliana plants. C.R. Acad. Sci. Paris Sciences de la vie, 1993,16:1194-1199.
Farran I, Sanchez-Serrano JJ, Medina JF, Prieto J, Mingo-Castel AM. Targeted expression of human serum albumin to potato tubers. Transgenic Res, 2002,1(4):337-46.
Goldschmidt-Clermont M. Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker for site-directed transformation of Chlamydomonas. Nucleic Acid Res.1991, 9: 4083-4089.
Gong XS and Yan LF. Improvement of chloroplast DNA isolation from higher plant. Science Bulletin. 1991, 6: 467-469.
Greiser-Wilke I., Moennig V., Coulibaly CO., Dahle J, Leder L. and Liess B. Identification of conserved epitopes on a hog cholera virus protein, Arch. Virol. 1990,111 (3-4): 213-225.
Hahn J, Park SH, Song JY, An SH and Ahn BY. Construction of recombinant swinepox viruses and expression of the classical swine fever E2 protein. J. Virol. Meth. 2001,93:49-56.
Hammond J M, McCoy R J, Jansen E S. 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. Vaccine ,2000,18 :1040~1050.
Han XQ, Liu XT, Zhang Y, Xie QG, Tian B. Study on the expression of E2 gene of classical swine fever virus in Pichia pastoris and the immunological activity of its expression product .Sheng Wu Gong Cheng Xue Bao. 2002,8(2):208-11.
Harris E.The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. San Diego: Academic Press, 1989,1-52
Hofgen R, Willmitzer L. Storage of competent cell for Agrobacterium transformation. Nucleic Acids Res, 1988,6:9877
Hulst M.M, Westra D.F, Wensvoort G and Moormann R.J. Glycoprotein El of hog cholera virus expressed in insect cells protects swine from hog cholera. J. Virol. 1993,67: 5435-5442.
Hulst MM, Himes G, Newbigin E and Moormann RJ. Glycoprotein E2 of classical swine fever virus: expression in insect cells and identification as a ribonuclease. Virology, 1994,10(2): 558-565.
Haydon D, Lea S, Fry L, et al. Characterizing sequence variation in the VP1 capsid proteins of foot and mouth disease virus (serotype 0) with respect to virion structure. J Mol Evo, 1998,46(4): 465-75.
Haq TA, Mason HS, Clements JD, et al. Oral immunization with a recombinant bacteriakl antigen produced in transgenic plants. Science, 1995,268:714-716.
Hein MB. Yeo TC, Wang F, Sturtevant A. Expression of cholera toxin subunits in plants. Ann NY Acad Sci. 1995,792:50-56.
Jani D, Singh NK, Bhattacharya S, et al. Studies on the immunogenic potential of plant-expressed cholera toxin B subunit. Plant Cell Rep, 2004,2 (7): 471-7.
Joung YH, Youm JW, Jeon JH, et al. Expression of the hepatitis B surface S and preS2 antigens in tubers of Solanum tuberosum. Plant Cell Rep, 2004,22(12): 925-30.
Konig M, Lengsfeld T, Pauly T. Classical swine fever virus: independent induction of protective immunity by two structural glycoproteins. J Virol, 1995,69(10):6479～6486.
Kindle KL, Richards KL, Stem DB. Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA. 1991,1721-1725.
Mason HS, Ball JM, Shi JS, Jiang X, Estes MK, Arntzen CJ: Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc Natl Acad Sci USA 1996, 93:5335-5340.
Markowska-Daniel I, Collins R.A and Pejsak Z. Evaluation of a genetic vaccine against classical swine fever. Vaccine, 2001,2480-2484.
Morilla-Gonzalez, A. A decade of learning to control CSF in endemic areas. In: Proceedings of the International of Pigs Veterinary Society, Ames, 2002,47-152.
Moennig V. Introduction to classical swine fever virus. Vet. Microbiol. 2000, 3:93-102.
Mercenier A, Wiedermann U and Breiteneder H. Edible genetically modified microorganisms and plants for improved health. Current Opinion in Biotechnology, 2001, 12:510-515.
Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant,1962, 5: 473-497. protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast. Biotechnol Lett, 2003,25(13): 1087-92.
Santos MJ, Wigdorovitz A, Trono K, et al. A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine, 2002,20(7-8): 1141-7
Sanchez J, Johansson S, Lowenadler B, Svennerholm AM, Holmgren J. Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination. Res Microbiol, 1990,141(7-8): 971-9.
Staub J, Garcia B, Hajdukiewicz P, Russell D, et al. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol. 2000,42:583-590.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press. 1989.
Stark, R., Rumenapf T, Meyers G, andThiel H.J. Genomic localization of hog cholera virus glycoproteins. Virology, 1990,174:286-289.
Terpstra C, Woortmeyer R and Barteling S.J. Development and properties of a cell culture produced vaccine for hog cholera based on the Chinese strain. Dtsch. Tierarztl. Wschr. 1990, 97: 77-79.
Tsibezov VV, Bogdanova VS, Zaberezhny AD, Grebennikova TV, Krivonos AV, Dudnikov LA, Kal'nov SL, Aliper TI, Nepoklonov EA. Use of recombinant protein E2 of the classical swine fever virus for immunization of animals. Vopr Virusol. 2000, 45(2): 36-41.
Van Regenmortel M.H.V, Fauquet C.M, Bishop D.H.L, Carstens E.B, Estes M.K, Lemon S.M, Maniloff M.A, Mayo D.J, McGeoch D.J, Pringle C.R. and Wickner R.B. Virus taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, Academic Press, San Diego, CA. 2000.
Van Oirschot JT. Vaccinology of classical swine fever: from lab to field. Vet Microbiol. 2003,96(4): 367-84
Van Rijn P.A., Bossers. A., Wensvoort. G. and Moormann. R.J. Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge. J. Gen. Virol. 1996,77: 2737-2745 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast. Biotechnol Lett, 2003,25(13): 1087-92.
Santos MJ, Wigdorovitz A, Trono K, et al. A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine, 2002,20(7-8): 1141-7
Sanchez J, Johansson S, Lowenadler B, Svennerholm AM, Holmgren J. Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination. Res Microbiol, 1990,141(7-8): 971-9.
Staub J, Garcia B, Hajdukiewicz P, Russell D, et al. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol. 2000,42:583-590.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press. 1989.
Stark, R., Rumenapf T, Meyers G, andThiel H.J. Genomic localization of hog cholera virus glycoproteins. Virology, 1990,174:286-289.
Terpstra C, Woortmeyer R and Barteling S.J. Development and properties of a cell culture produced vaccine for hog cholera based on the Chinese strain. Dtsch. Tierarztl. Wschr. 1990, 97: 77-79.
Tsibezov VV, Bogdanova VS, Zaberezhny AD, Grebennikova TV, Krivonos AV, Dudnikov LA, Kal'nov SL, Aliper TI, Nepoklonov EA. Use of recombinant protein E2 of the classical swine fever virus for immunization of animals. Vopr Virusol. 2000, 45(2): 36-41.
Van Regenmortel M.H.V, Fauquet C.M, Bishop D.H.L, Carstens E.B, Estes M.K, Lemon S.M, Maniloff M.A, Mayo D.J, McGeoch D.J, Pringle C.R. and Wickner R.B. Virus taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, Academic Press, San Diego, CA. 2000.
Van Oirschot JT. Vaccinology of classical swine fever: from lab to field. Vet Microbiol. 2003,96(4): 367-84
Van Rijn P.A., Bossers. A., Wensvoort. G. and Moormann. R.J. Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge. J. Gen. Virol. 1996,77: 2737-2745
Van Zijl. M, Wensvoort G, De Kluyver E, Hulst M, Van der Gulden H, Gielkens A, Berns A and Moormann R. Live attenuated pseudorabies virus expressing envelope glycoprotein El of hog cholera virus protects swine against both pseudorabies and hog cholera. J. Virol. 1991, 65: 2761-2765.
Wandelt CI, Khan MR, Craig S, Schroeder HE, Spencer D, Higgins TJ. Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants. Plant J, 1992, 2(2): 181-92
Wigdorovitz A, Carrillo C, Dus-Santos MJ, et al. Induction of a protective antibody response to foot-and-mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1. Virology, 1999,255:347-353.
Walmsley AM, Arntzen CJ. Plants for delivery of edible vaccines.Curr Opin Biotechnol, 2000,11:126-129.
Walmsley AM and Arntzeny CJ. Plant cell factories and mucosal vaccines, Current Opinion in Biotechnology, 2003,14:145-150
Wensvoort G. Epitopes on structural proteins of hog cholera (swine fever) virus. Ph.D. Thesis. Utrecht University. 1989.
Wong ML, Liu JJ, Chang Y, Chang TJ.Expression of the glycoprotein E2 of the classical swine fever virus in Escherichia coli. 1: J Vet Med Sci. 1998,60(4): 541 -4
Zhang YG, Liu XT, Han XQ, Liu XC, Zhang YM, Xie QG. Cloning of the major antigen region of E2 gene of hog cholera virus and expression in Escherichia coli. Sheng Wu Gong Cheng Xue Bao. 2002,18(5): 605-8.
Zheng Z X, Xu Q X. Recombinant and synthetic vaccines: Biosynthetic peptide vaccine against foot-and-mouth disease virus .New Deli India: Naruse Publishing House, 1994. 30-35.
Zora S and Maliga P. High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA. 1993, 90: 913-917
ZHANG Y G, LIU X T, HAN X Q, et al. Cloning of the major antigen region of E2 gene of hog choleravirus and expression in Escherichia coli. Chinese Journal of Biotechnology, 2002,18(5) : 605 - 608
胡建和,陈永耀,王自良,谢庆阁.新型猪瘟疫苗的研究和应用前景.中国兽医科技.2003,33:36-39.
胡建和,张彦明,谢庆阁.猪瘟病毒的分子生物学研究进展.动物医学进展.2002,23:15-18.
J.萨姆布鲁克,D.W.拉塞尔.分子克隆实验指南.第三版.北京:科学出版社,2002.27
蒋文俊,李华春,李乐,信爱国,廖德芳 口蹄疫疫苗研究进展 畜牧与兽医,2004,36:42-43.
刘晓松.家畜口蹄疫研究概况及防制措施.内蒙古畜牧科学,2000,21(1):46—48.
刘思国.猪瘟病毒 E2.蛋白抗原表位的研究博士后研究报告.2001,长春.
孔桂美.应用植物表达系统生产疫苗的研究概况.生物技术通.2002,13:226-227.
任志强,刘惠民,李润植,杨慧珍.转基因植物作为生物反应器在疫苗生产中的应用.中国生物工程杂志,2003,23:31-35.
苏宁,沈桂芳.植物基因工程新途径:叶绿体转化 生物技术通报,2001,4:9-13.
杨永钦,李乐.口蹄疫灭活二联苗免疫研究.中国畜禽传染病,1995,82(3):29—30.
尹德华,韩福祥.家畜口蹄疫及其防制.北京:中国农业出版社,1994.
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.中国病毒学,2000,15(3):264～271.
赵启祖,谢庆阁.家畜口蹄疫疫苗简介.中国兽医技术,2000,30(6):43—44.
赵凯,邹仕平,张震宇,等.抗O型口蹄疫病毒DNA疫苗的建立.中国免疫学杂志,2002,18(2):89—92.
赵耘,宁宜宝.猪瘟疫苗研究进展.动物医学进展.2004,25(1):39-42.
周鹏程,陆宇,陈建国.猪瘟病毒E2(gp55)基因的克隆表达及其DNA疫苗的初 步研究.微生物学报,2000,40(3):223～251.
Andrew ME, Morrissy CJ, Lenghaus C, Oke PG, Sproat K.W., Hodgson ALM, Johnson M. A and Coupa BEH.Protection of pigs against classical swine fever with DNA-delivered gp55. Vaccine, 2000, 8: 1932-1938.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 2: 248-54.
Bouma A, De Smit A.J, De Kluijver E.P, Terpstra C and Moormann R. J. Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus. Vet. Microbiol. 1999, 66(2): 101-114.
Barteling SJ, Vreeswijk J. Development in foot-and-mouth disease vaccines. Vaccine, 1992,75-88.
Bittle JL, Houghton PA, Alexancler H, et al. Protection against foot-and-mouth disease by immunization with a Chemically Synthesized peptide predicted from the viral nucleotide sequence. Nature, 1982, 298:30-33.
Brown F. Vaccination against foot and mouth disease virus. Vaccine, 1992, 10:1022-1026.
Brown F. New approaches to vaccine against foot-and-mouth disease. Vaccine, 1988,6:180-183.
Blowers AD.Transcriptional analysis of endogenous and foreign genes in chloroplast transformants of Chlamydomonas. Plant Cell, 1990, 2:1059-1070.
Boynton JE, Gillham NW, Harris EH, et al. Chloroplast transformation inChlamydomonas with high velocity microprojectiles. Science, 1988, 240: 1534-1538.
Carrillo C, Wigdorovitz A, Oliveros JC, et al. Protective immune response tofoot-and-mouth disease virus with VP1 expressed in transgenic plants. J Virol. 1998,72:1688-1690.
Carrillo C, Wigdorovitz A, Trono K, et al. Induction of a virus-specific antibody response to foot and mouth disease virus using the structural protein VP1 expressed in transgenic potato plants. Viral Immunol, 2001,14(1): 49-57
Curtiss RI, Cardineau CA. Oral immunization by transgenic plants. World Patent Application ,1990, WO 90/02484.
Daniell, H. New tools for chloroplast genetic engineering. Nal Biotechnol. 1999,17:855-856.
Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: production of antibodies biopharmaceuticals and edible vaccine in plants. Trends in plant Science. 2001,6:219-226.
Daniell H, Muthukumar B, Lee SB. Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection. Curr Genet. 2001, 39:109-116.
De Cosa B, Moar W, Lee SB, Miller M, Daniell H. Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol. 2001,19:71-74.
Dus Santos MJ, Wigdorovitz A, Trono K, et al. A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants.Vaccine, 2002,20(7-8): 1141-7.
Davies G. Foot and mouth disease. Res Vet Sc, 2002,3(3): 195-9.
De Smit A.J, Bouma A, De Kluijver E.P, Terpstra C and Moormann R.J. Duration ofn the protection of an E2 subunit marker vaccine against classical swine fever after a single vaccination. Vet. Microbiol. 2001, 8 : 307-317.
Depner K.R, Bouma A, Koenen F, Klinkenberg D, Lange E, De Smit J.A and Vanderhallen H. Classical swine fever (CSF) marker vaccine. Trial II. Challenge study in pregnant sows. Vet. Microbiol. 2001, 83: 107-120.
Dong XN, Wei K, Liu ZQ, Chen YH. Candidate peptide vaccine induced protection against classical swine fever virus. Vaccine. 2002, (3-4): 167-73.
Edwards S, Fukusho A and Lefevre P.C. Classical swine fever: the global situation. Vet. Microbiol 2000, 3(2-3): 103-119.
Echtold, N., J. Ellis, and G. Pelletier. Agrobacterium mediated gene transfer by infiltration of adult Arabidpsis thaliana plants. C.R. Acad. Sci. Paris Sciences de la vie, 1993,16:1194-1199.
Farran I, Sanchez-Serrano JJ, Medina JF, Prieto J, Mingo-Castel AM. Targeted expression of human serum albumin to potato tubers. Transgenic Res, 2002,1(4):337-46.
Goldschmidt-Clermont M. Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker for site-directed transformation of Chlamydomonas. Nucleic Acid Res.1991, 9: 4083-4089.
Gong XS and Yan LF. Improvement of chloroplast DNA isolation from higher plant. Science Bulletin. 1991, 6: 467-469.
Greiser-Wilke I., Moennig V., Coulibaly CO., Dahle J, Leder L. and Liess B. Identification of conserved epitopes on a hog cholera virus protein, Arch. Virol. 1990,111 (3-4): 213-225.
Hahn J, Park SH, Song JY, An SH and Ahn BY. Construction of recombinant swinepox viruses and expression of the classical swine fever E2 protein. J. Virol. Meth. 2001,93:49-56.
Hammond J M, McCoy R J, Jansen E S. 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. Vaccine ,2000,18 :1040~1050.
Han XQ, Liu XT, Zhang Y, Xie QG, Tian B. Study on the expression of E2 gene of classical swine fever virus in Pichia pastoris and the immunological activity of its expression product .Sheng Wu Gong Cheng Xue Bao. 2002,8(2):208-11.
Harris E.The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. San Diego: Academic Press, 1989,1-52
Hofgen R, Willmitzer L. Storage of competent cell for Agrobacterium transformation. Nucleic Acids Res, 1988,6:9877
Hulst M.M, Westra D.F, Wensvoort G and Moormann R.J. Glycoprotein El of hog cholera virus expressed in insect cells protects swine from hog cholera. J. Virol. 1993,67: 5435-5442.
Hulst MM, Himes G, Newbigin E and Moormann RJ. Glycoprotein E2 of classical swine fever virus: expression in insect cells and identification as a ribonuclease. Virology, 1994,10(2): 558-565.
Haydon D, Lea S, Fry L, et al. Characterizing sequence variation in the VP1 capsid proteins of foot and mouth disease virus (serotype 0) with respect to virion structure. J Mol Evo, 1998,46(4): 465-75.
Haq TA, Mason HS, Clements JD, et al. Oral immunization with a recombinant bacteriakl antigen produced in transgenic plants. Science, 1995,268:714-716.
Hein MB. Yeo TC, Wang F, Sturtevant A. Expression of cholera toxin subunits in plants. Ann NY Acad Sci. 1995,792:50-56.
Jani D, Singh NK, Bhattacharya S, et al. Studies on the immunogenic potential of plant-expressed cholera toxin B subunit. Plant Cell Rep, 2004,2 (7): 471-7.
Joung YH, Youm JW, Jeon JH, et al. Expression of the hepatitis B surface S and preS2 antigens in tubers of Solanum tuberosum. Plant Cell Rep, 2004,22(12): 925-30.
Konig M, Lengsfeld T, Pauly T. Classical swine fever virus: independent induction of protective immunity by two structural glycoproteins. J Virol, 1995,69(10):6479～6486.
Kindle KL, Richards KL, Stem DB. Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA. 1991,1721-1725.
Mason HS, Ball JM, Shi JS, Jiang X, Estes MK, Arntzen CJ: Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc Natl Acad Sci USA 1996, 93:5335-5340.
Markowska-Daniel I, Collins R.A and Pejsak Z. Evaluation of a genetic vaccine against classical swine fever. Vaccine, 2001,2480-2484.
Morilla-Gonzalez, A. A decade of learning to control CSF in endemic areas. In: Proceedings of the International of Pigs Veterinary Society, Ames, 2002,47-152.
Moennig V. Introduction to classical swine fever virus. Vet. Microbiol. 2000, 3:93-102.
Mercenier A, Wiedermann U and Breiteneder H. Edible genetically modified microorganisms and plants for improved health. Current Opinion in Biotechnology, 2001, 12:510-515.
Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant,1962, 5: 473-497. protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast. Biotechnol Lett, 2003,25(13): 1087-92.
Santos MJ, Wigdorovitz A, Trono K, et al. A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine, 2002,20(7-8): 1141-7
Sanchez J, Johansson S, Lowenadler B, Svennerholm AM, Holmgren J. Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination. Res Microbiol, 1990,141(7-8): 971-9.
Staub J, Garcia B, Hajdukiewicz P, Russell D, et al. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol. 2000,42:583-590.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press. 1989.
Stark, R., Rumenapf T, Meyers G, andThiel H.J. Genomic localization of hog cholera virus glycoproteins. Virology, 1990,174:286-289.
Terpstra C, Woortmeyer R and Barteling S.J. Development and properties of a cell culture produced vaccine for hog cholera based on the Chinese strain. Dtsch. Tierarztl. Wschr. 1990, 97: 77-79.
Tsibezov VV, Bogdanova VS, Zaberezhny AD, Grebennikova TV, Krivonos AV, Dudnikov LA, Kal'nov SL, Aliper TI, Nepoklonov EA. Use of recombinant protein E2 of the classical swine fever virus for immunization of animals. Vopr Virusol. 2000, 45(2): 36-41.
Van Regenmortel M.H.V, Fauquet C.M, Bishop D.H.L, Carstens E.B, Estes M.K, Lemon S.M, Maniloff M.A, Mayo D.J, McGeoch D.J, Pringle C.R. and Wickner R.B. Virus taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, Academic Press, San Diego, CA. 2000.
Van Oirschot JT. Vaccinology of classical swine fever: from lab to field. Vet Microbiol. 2003,96(4): 367-84
Van Rijn P.A., Bossers. A., Wensvoort. G. and Moormann. R.J. Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge. J. Gen. Virol. 1996,77: 2737-2745 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast. Biotechnol Lett, 2003,25(13): 1087-92.
Santos MJ, Wigdorovitz A, Trono K, et al. A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine, 2002,20(7-8): 1141-7
Sanchez J, Johansson S, Lowenadler B, Svennerholm AM, Holmgren J. Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination. Res Microbiol, 1990,141(7-8): 971-9.
Staub J, Garcia B, Hajdukiewicz P, Russell D, et al. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol. 2000,42:583-590.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press. 1989.
Stark, R., Rumenapf T, Meyers G, andThiel H.J. Genomic localization of hog cholera virus glycoproteins. Virology, 1990,174:286-289.
Terpstra C, Woortmeyer R and Barteling S.J. Development and properties of a cell culture produced vaccine for hog cholera based on the Chinese strain. Dtsch. Tierarztl. Wschr. 1990, 97: 77-79.
Tsibezov VV, Bogdanova VS, Zaberezhny AD, Grebennikova TV, Krivonos AV, Dudnikov LA, Kal'nov SL, Aliper TI, Nepoklonov EA. Use of recombinant protein E2 of the classical swine fever virus for immunization of animals. Vopr Virusol. 2000, 45(2): 36-41.
Van Regenmortel M.H.V, Fauquet C.M, Bishop D.H.L, Carstens E.B, Estes M.K, Lemon S.M, Maniloff M.A, Mayo D.J, McGeoch D.J, Pringle C.R. and Wickner R.B. Virus taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, Academic Press, San Diego, CA. 2000.
Van Oirschot JT. Vaccinology of classical swine fever: from lab to field. Vet Microbiol. 2003,96(4): 367-84
Van Rijn P.A., Bossers. A., Wensvoort. G. and Moormann. R.J. Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal CSFV challenge. J. Gen. Virol. 1996,77: 2737-2745
Van Zijl. M, Wensvoort G, De Kluyver E, Hulst M, Van der Gulden H, Gielkens A, Berns A and Moormann R. Live attenuated pseudorabies virus expressing envelope glycoprotein El of hog cholera virus protects swine against both pseudorabies and hog cholera. J. Virol. 1991, 65: 2761-2765.
Wandelt CI, Khan MR, Craig S, Schroeder HE, Spencer D, Higgins TJ. Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants. Plant J, 1992, 2(2): 181-92
Wigdorovitz A, Carrillo C, Dus-Santos MJ, et al. Induction of a protective antibody response to foot-and-mouth disease virus in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1. Virology, 1999,255:347-353.
Walmsley AM, Arntzen CJ. Plants for delivery of edible vaccines.Curr Opin Biotechnol, 2000,11:126-129.
Walmsley AM and Arntzeny CJ. Plant cell factories and mucosal vaccines, Current Opinion in Biotechnology, 2003,14:145-150
Wensvoort G. Epitopes on structural proteins of hog cholera (swine fever) virus. Ph.D. Thesis. Utrecht University. 1989.
Wong ML, Liu JJ, Chang Y, Chang TJ.Expression of the glycoprotein E2 of the classical swine fever virus in Escherichia coli. 1: J Vet Med Sci. 1998,60(4): 541 -4
Zhang YG, Liu XT, Han XQ, Liu XC, Zhang YM, Xie QG. Cloning of the major antigen region of E2 gene of hog cholera virus and expression in Escherichia coli. Sheng Wu Gong Cheng Xue Bao. 2002,18(5): 605-8.
Zheng Z X, Xu Q X. Recombinant and synthetic vaccines: Biosynthetic peptide vaccine against foot-and-mouth disease virus .New Deli India: Naruse Publishing House, 1994. 30-35.
Zora S and Maliga P. High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA. 1993, 90: 913-917
ZHANG Y G, LIU X T, HAN X Q, et al. Cloning of the major antigen region of E2 gene of hog choleravirus and expression in Escherichia coli. Chinese Journal of Biotechnology, 2002,18(5) : 605 - 608