人源轮状病毒在胡萝卜中的表达
摘要
本实验利用胡萝卜作为生物反应器受体生产轮状病毒外壳蛋白VP6,VP7。通过PCR,RT-PCR,Western Blot,ELISA检测整合有轮状病毒外源基因的胡萝卜植株,即从基因水平到蛋白水平逐步检测。并将蛋白水平上表达有轮状病毒外壳基因的胡萝卜植株免疫小鼠,免疫方法采用注射免疫和灌胃免疫。
1提取胡萝卜DNA进行PCR分析,表明VP6,VP7,VP7-U基因都已整合到胡萝卜植株基因组中。
2转基因植株的RT-PCR分析显示部分植株进行了反转录。
3对转录水平上有表达的转基因植株进行Western Blot和ELISA分析,结果表明转基因胡萝卜植株可以有效地表达VP7蛋白,并具有一定的免疫原性。
4利用转VP7基因胡萝卜根部蛋白注射免疫小鼠,抗原免疫量为1μg左右,免疫周期为两周,免疫三次后采血并提取小鼠血清进行ELISA分析,结果表明注射阳性胡萝卜植株蛋白的小鼠体内IgG含量要高于注射阴性胡萝卜植株蛋白的小鼠及未免疫的小鼠。利用转VP7-U基因胡萝卜根部蛋白灌胃免疫小鼠,抗原量为20-30μg,免疫周期为一周,免疫四次后采血提取血清并进行ELISA检测,结果表明灌服VP7-U基因的胡萝卜植株蛋白的小鼠IgG含量要高于灌服阴性植株的小鼠或未灌服小鼠的IgG的含量。
5同时将转VP7-U基因的植株抗原量与VP7基因植株抗原量进行比较,ELISA分析表明,转VP7-U基因的植株抗原量要高于VP7基因植株抗原量。
Carrot was applied in the experiment as receptor of bioreactor to produce RV protein VP6, VP7. And transgenic carrots in different generations were screened with the methods of PCR,RT-PCR,Western Blot,ELISA. The transgenic carrots expressing RV protein were applied to immune mice.
1 The PCR analysis showed that VP6,VP7,VP7-U gene had integrated to carrot genome.
2 The result of RT-PCR demonstrated that only some transgenic carrots had reverse transcriptase procedure.
3 Western Blot and ELISA showed that VP7 protein had been expressed effectively in transgenic carrots and the product had a certain immunogenicity.
4 The root protein of transgenic carrot with VP7 gene was applied to immune mice through injection together with freund's adjuvant and the dosage of antigen was about 1μg. The immunization period was two weeks and mice blood was got to extract serum after three times immunization. The ELISA analysis for serum showed that the content of IgG in the mice injecting positive carrot root protein was higher than that of the mice injecting negative carrot root protein or non. The root protein of transgenic carrot with VP7-U gene was extracted to immune mice through filling stomach after analysises of PCR, RT-PCR,Western Blot,ELISA,and the antigen dosage was 20μg to 30μg. The immunization period was one week and mice blood was got to extract serum after four times immunization for ELISA analysis. The ELISA result showed that the content of IgG in the mice injecting positive carrot root protein was higher than that of the mice injecting negative carrot root protein or non.
5 Comparing with the carrots transformed VP7 gene, the antigen content in carrots transformed VP7-U gene was higher.
1提取胡萝卜DNA进行PCR分析,表明VP6,VP7,VP7-U基因都已整合到胡萝卜植株基因组中。
2转基因植株的RT-PCR分析显示部分植株进行了反转录。
3对转录水平上有表达的转基因植株进行Western Blot和ELISA分析,结果表明转基因胡萝卜植株可以有效地表达VP7蛋白,并具有一定的免疫原性。
4利用转VP7基因胡萝卜根部蛋白注射免疫小鼠,抗原免疫量为1μg左右,免疫周期为两周,免疫三次后采血并提取小鼠血清进行ELISA分析,结果表明注射阳性胡萝卜植株蛋白的小鼠体内IgG含量要高于注射阴性胡萝卜植株蛋白的小鼠及未免疫的小鼠。利用转VP7-U基因胡萝卜根部蛋白灌胃免疫小鼠,抗原量为20-30μg,免疫周期为一周,免疫四次后采血提取血清并进行ELISA检测,结果表明灌服VP7-U基因的胡萝卜植株蛋白的小鼠IgG含量要高于灌服阴性植株的小鼠或未灌服小鼠的IgG的含量。
5同时将转VP7-U基因的植株抗原量与VP7基因植株抗原量进行比较,ELISA分析表明,转VP7-U基因的植株抗原量要高于VP7基因植株抗原量。
Carrot was applied in the experiment as receptor of bioreactor to produce RV protein VP6, VP7. And transgenic carrots in different generations were screened with the methods of PCR,RT-PCR,Western Blot,ELISA. The transgenic carrots expressing RV protein were applied to immune mice.
1 The PCR analysis showed that VP6,VP7,VP7-U gene had integrated to carrot genome.
2 The result of RT-PCR demonstrated that only some transgenic carrots had reverse transcriptase procedure.
3 Western Blot and ELISA showed that VP7 protein had been expressed effectively in transgenic carrots and the product had a certain immunogenicity.
4 The root protein of transgenic carrot with VP7 gene was applied to immune mice through injection together with freund's adjuvant and the dosage of antigen was about 1μg. The immunization period was two weeks and mice blood was got to extract serum after three times immunization. The ELISA analysis for serum showed that the content of IgG in the mice injecting positive carrot root protein was higher than that of the mice injecting negative carrot root protein or non. The root protein of transgenic carrot with VP7-U gene was extracted to immune mice through filling stomach after analysises of PCR, RT-PCR,Western Blot,ELISA,and the antigen dosage was 20μg to 30μg. The immunization period was one week and mice blood was got to extract serum after four times immunization for ELISA analysis. The ELISA result showed that the content of IgG in the mice injecting positive carrot root protein was higher than that of the mice injecting negative carrot root protein or non.
5 Comparing with the carrots transformed VP7 gene, the antigen content in carrots transformed VP7-U gene was higher.
引文
[1]周俊辉,周家,曾浩森,等.园艺植物组织培养中的褐化现象及抗褐化研究进展[J].园艺学报,2000,27(51):481-486.
[2]张淑红,王蒂,王清.影响马铃薯叶肉原生质体褐化的因素及AgNO3对其褐化和分裂的作用[J].中国马铃薯,2004,18(2):77-81.
[3]何松林,陈笑蕾,陈莉,等.牡丹叶柄离体培养中褐化防止的初步研究[J].河南科学,2005,23(1):47-50.
[4]郝林,徐昕,王尊生.重组蛋白在植物体中的表达及其应用[J].植物学通报,2004,21(1):2101-2112.
[5] Streatfield SJ,Howard JA.Plant production systems for vaccines[J].Expert Rev Vaccines. 2003,2(6):763-775.
[6] Fischer R,Hoffmann K,Schillberg S,et al.Antibody production by molecular farming in plants[J].Bio Regul Homeost Agent 2000,14(2):83-92.
[7] Twyman R M,Stoger E,Schillberg S,et al.Molecular farming in plants[J].Host systems and expression technology[J].Trends Biotechnol,2003,22(12):570-578.
[8] Mason H S,Lam DMK,Amtzen CJ.Expression of hepatitis B surface antigen in transgenic plants[J].Proc Natl Acad Sci USA,1992,89:11745-11749.
[9] Tregoning JS,Nixon P,Kur oda H,et al.Expression of tetanus toxin Fragment Cin tobacco chlorphast[J].Nucleic Acids Res,2003,31(4):1174-1179.
[10] Tacket CO,Mason HS,Loson G,et al.Human immune response to a novel Nor walk virus vaccine delivered in transgenic potatoes[J].Infect Dis,2000,182(1):302-305.
[11] Mason HS,Warzecha H,Mor T,et al.Edible plant vaccines:applications for prophylactic and therapeutic molecular medicine[J].Trends Mol Med.2002,8(7):324-329.
[12] Ma JK,Drake PM,Christou P.The production of recombinant pharmaceutical proteins in plants[J].Nat Rev Genet.2003,4(10):794-805.
[13] Drake PM,Chargfelegue D,Vine ND,et al.Transgenic plants expressing antibodies:a model for phytoremediation[J].FASEB J.2002,16(14):1855-1860.
[14] Ma JK,Vine ND.Plant expression systems for the production of vaccines[J].Curr Top Microbiol Immunol.1999,236:275-292.
[15]杨振泉,刘巧泉,焦新安.利用转基因植物表达药用蛋白[J].中国生物工程杂志,2004,24(3):22-25.
[16]江小玲,贺竹梅,俞守义等.转基因植物口服疫苗的安全性问题所引发的思考[J].医学与社会,2004,17(2):17-18.
[17]苏钢.转基因蔬菜口服疫苗研究进展[J].邢台师范高专学报,2002,17(2):65-67.
[18]马三梅,王永飞,王亚琴.利用转基因植物生产可食疫苗[J].生命的化学,2004,24(1):22-24.
[19]刘红莉,雷霆,王一理.转基因植物疫苗的研究策略[J].中国生物工程杂志,2004,24(4):30-33.
[20] Yu J,Langridge W H.A plant-based multicomponent vaccine protects mice from enteric diseases[J].Nat Biotechnol,2001,19:548-552.
[21] Herman EM,Melroy DL,Buckhout TJ(1990) Apparent processing of a soybean oil bodymembrane protein accompanies the onset of oil mobilization.Plant Physiol 94: 341-349.
[22] Ma J K,Hiatt A,Hein M,et al.Generation and assembly of secretory antibodies in plants[J].Science,1995,286(5211):716-719.
[23]耿德贵,王义琴,李文彬等.利用转基因植物生产口服疫苗的研究现状[J].生物工程进展,2002,22(1):19-210.
[24]李轶女,胡英考.转基因植物基因工程疫苗[J].生物技术通报,2002,2:11-15.
[25]凌华,黄惠琴,鲍时翔.植物生物反应器研究进展[J].中国生物工程杂志,2002,22(5):21-25.
[26]任志强,刘惠民,李润植等.转基因植物作为生物反应器在疫苗生产中的应用[J].中国生物工程杂志,2003,23(6):31-35.
[27]王瑞刚,王水平.外源蛋白表达系统及利用植物表达外源蛋白的特点与优势[J].生物学通报,2003,38(1):11-12.
[28] Giddings G,Allison G,Brooks D,et al,Transgenic plants as factories for biopharmaceutical[J].Nat Biotechno1,2000(18):1151-1155.
[29] Twyman RM,Stoger E,Schillberg S,Christou P,Fischer R (2003) Molecular farming in plants: host systems and expression technology.Trends Biotechnol 21:570-578.
[30] Haq T A,Mason H S,Clements J D,et al.Oral immunization with recombinant bacterial antigen produced in transgenic plants.Science,1995,268:714-716.
[31] Mason H S,Haq T A,Clements J D,et al.Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin(LT):potatoes expressing a synthetic LT-B gene.Vaccine,1998, 16:1336-1343.
[32] Streatfield S J,Jilka J M,Hood E E,et al.Plant-based vaccines:unique advantages.Vaccine, 2001,19:2742-2748.
[33] Arakawa T,Chong D K X,Langridge W H R.Efficacy of a food plant based oral cholera toxin B subunit vaccine.Nature Biotechnol,1998,16:292-297.
[34] Jani D,Singh N K,Bhattacharya S,et al.Studies on the immunogenic potential of plant- expressed cholera toxin B subunit.Plant Cell Rep,2004,22:471-477.
[35] Mason H S,Ball J M,Shi J J,et al.Experssion of Nowalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.Proc Natl Acad Sci USA,1996,93:5335-5340.
[36] SandhuJ S,Krasnyanski S F,Dormer L L,et al.Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-F Protein induces a systemic immune response.Transgenic Res,2000,9:127-135.
[37] Muller C P,Fack F D,Damien et al.Immunogenic measles antigens expressed in plants: role as an Edible vaccine for adults.Vaccine,2003,21:816-819.
[38] Khandelwal A,Renukaradhya G J,Rajasekhar M,et al.Systemic and oral immunogenicity of hemagglutinin protein of rinderpestvirus expressed by transgenic peanut plants in a mouse model.Virology,2004,323:284-291.
[39] Karasev A V,Foulke S,Wellens C,et al.Plant based HIV-l vaccine candidate:Tat protein produced in spinach.Vaccine,2005,23:1875-1880.
[40] Arakawa T,Yu J,Langridge W H R.Synthesis of a cholera toxin B subunit-rotavirus NSP4fusion protein in potato.Plant Cell Rep,2001,20:343-348.
[41] Kim T G,Langridge W H R.Synthesis of an HIV-1Tat transduction domain-rotavius enterotoxin Fusion protein transgenic potato.Plant Ceill Rep,2004,22:382-387.
[42] Wigdorovitz A,Mozgovoj M,Dus Santos M J et al.Protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants[J].Gen.Virol, 2004,85:1825-1832.
[43] Perez Filgueira D M,Mozgovoj M,Wigdorovitz A,et al.Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8* produced in plants using a TMV-based vector.Arch Virol,2004,149:2337-2348.
[44] Matsumura T,Itchoda N,Tsunemitsu H.Porduction of immunogenic VP6 protein of bovine group A rotavirus in transgenic potato plants.Arch Virol,2002,147:1263-1270.
[45] Yu J,Langridge W.Expression of rotavirus capsid protein VP6 in transgenic potato and its oral immunogenicity in mice.Transgenic Res,2003,12:163-169.
[46] Wu Y Z,Li J T,Mou Z R,et al.Immunization with rotavirus VP7 expressed in transgenic potatoes induced high titers of mucosal neutralizing IgA.Virology,2003,313:337-342.
[47] Bishop R.R.,Davidson G.P.,Holmes I. H.et al.Virus Particles in epithelial cells of duodena mucosa from children with acute non-bacterial gastroenteritis.Lancet.1973,2:1281-1283.
[48] ESTES M K,COHEN J.Rotavirus gene structure and function[J].Microbiol Rev,1989, 53(4) :410-419.
[49] Institute of Medicine.The prospects for immunizing against rotavirus in new vaccine development.Establishing Priorities.Diseases of importance in developing countries.VOlII Washington,DC:National Academy Press,1986,318-388.
[50]王春凤,丛彦龙.轮状病毒研究现状[J].人畜共患传染病防治研究新成果汇编.
[51] Kalica AR,Flores J,Greenberg HB.Identification of the rotaviral gene that codes for hemagglutinin and protease-enhanced plaque formation.Virol.1983,125:194-205.
[52] Davidson G. P,Goller I,Bishop R. F.et al.Immunoflunrescence in duodenal mucosa of children with acute enteritis due to a new virus[J].Clin.Pathol.1975,28:263-266.
[53] Greenberg H.G.Clack H.E.Offit P.A.Rotavirus pathology and pathophysiology.Curr.Top. Microbiol.Immunol.1994.185:255-283.
[54] Stephen. J.Functional abnormalities in intestine.In“Viruses and the Gut”,proceedings of the Ninth BSG SKSF International Workshop(M.J.G. Farthing,Ed.) PP.41-44.Swan Press. London,1989.
[55] Del Castillo J.R.,Ludert J.E,Sanchez A.et al.Rotavirus in fection alters Na+ and K+ homeostasis in MA-104 cells[J].Gen.Virol.1991,72:541-547.
[56] Michelangeli F.Ruiz M.C.Del Castillo J.R.,et al.Effect of rotavirus infection on Intracellular calcium homeostasis in cultured cells.Virology,1991,181:520-527.
[57] Tian P.Ester M.K.,HuY.et al.The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca from the endoplasmic reticulum[J].Virol.1995,69:5763-5772.
[58] Ward L.A.,Rosen B.L.Yuan L.et al.Pathogenesis of an attenuated and a virulent strain of group A human rotavirus in neonatal gnotobiotic pigs[J].Gen.Virol,1996,77:1431-1441.
[59] Eglee Perez M,Glass R,Alvarez G,Pericchi LR,Gonzalez R,et al.(2000) Rhesusrotavirus-based quadrivalent vaccine is efficacious despite age,socioeconomic conditions and seasonality in Venezuela.Vaccine 19:976-981.
[60] Estes MK,Conner ME,Gilger MA,Graham DY(1989) Molecular biology and immunology of rotavirus infections.Immunol Invest 18:571-581.
[61] Affranchino J.L.,Pollevick,G.and Frasch,A.C.C.(1991)The expression of the major shedTrypanosoma cruzi antigen results from the developmentally-regulated transcription of a small gene family.FEBS Lett 2:316-320.
[62] McNeal MM,VanCott JL,Choi AH,Basu M,Flint JA,et al.(2002) CD4 T cells are the only lymphocytes needed to protect mice against rotavirus shedding after intranasal immunization with a chimeric VP6 protein and the adjuvant LT(R192G)[J].Virol 76:560-568.
[63] Choi NW,Estes MK,Langridge WH (2005) Oral immunization with a shiga toxin B subunit: rotavirus NSP4(90) fusion protein protects mice against gastroenteritis.Vaccine 23: 5168-5176.
[64] Herrmann JE,Chen SC,Fynan EF,Santoro JC,Greenberg HB,et al.(1996) Protection against rotavirus infections by DNA vaccination. J Infect Dis 174 Suppl 1:93-97.
[65] Steele AD,Basetse HR,Blacklow NR,Herrmann JE(1998) Astrovirus infection in South Africa: a pilot study.Ann Trop Paediatr 18:315-319.
[66] Xu WC,Chen BT,Fu JX(1997) [Progress in the study on the treatment of rotaviral enteritis with traditional Chinese medicine].Zhongguo Zhong Xi Yi Jie He Za Zhi 17:511-512.
[67] Chen HN,Dennehy PH,Oh W,Lee CN,Huang ML,et al.(1997) Outbreak and control of a rotaviral infection in a nursery[J].Formos Med Assoc 96:884-889.
[68] Chen SC,Fynan EF,Robinson HL,Lu S,Greenberg HB,et al.(1997) Protective immunity induced by rotavirus DNA vaccines.Vaccine 15:899-902.
[69] Mat sumura T,Itchoda N,Tsunemtsu H.Production of immunogenic VP6 protein of bovine group A rotavirus in transgenic potato plants[J].Arch Virol,2002,147:1263-1270.
[70] Hardegger M,Sturm A.Transformation and regeneration of carrot (Daucus Carota L.)[J].Molecular Breeding,1998,4:119-127.
[2]张淑红,王蒂,王清.影响马铃薯叶肉原生质体褐化的因素及AgNO3对其褐化和分裂的作用[J].中国马铃薯,2004,18(2):77-81.
[3]何松林,陈笑蕾,陈莉,等.牡丹叶柄离体培养中褐化防止的初步研究[J].河南科学,2005,23(1):47-50.
[4]郝林,徐昕,王尊生.重组蛋白在植物体中的表达及其应用[J].植物学通报,2004,21(1):2101-2112.
[5] Streatfield SJ,Howard JA.Plant production systems for vaccines[J].Expert Rev Vaccines. 2003,2(6):763-775.
[6] Fischer R,Hoffmann K,Schillberg S,et al.Antibody production by molecular farming in plants[J].Bio Regul Homeost Agent 2000,14(2):83-92.
[7] Twyman R M,Stoger E,Schillberg S,et al.Molecular farming in plants[J].Host systems and expression technology[J].Trends Biotechnol,2003,22(12):570-578.
[8] Mason H S,Lam DMK,Amtzen CJ.Expression of hepatitis B surface antigen in transgenic plants[J].Proc Natl Acad Sci USA,1992,89:11745-11749.
[9] Tregoning JS,Nixon P,Kur oda H,et al.Expression of tetanus toxin Fragment Cin tobacco chlorphast[J].Nucleic Acids Res,2003,31(4):1174-1179.
[10] Tacket CO,Mason HS,Loson G,et al.Human immune response to a novel Nor walk virus vaccine delivered in transgenic potatoes[J].Infect Dis,2000,182(1):302-305.
[11] Mason HS,Warzecha H,Mor T,et al.Edible plant vaccines:applications for prophylactic and therapeutic molecular medicine[J].Trends Mol Med.2002,8(7):324-329.
[12] Ma JK,Drake PM,Christou P.The production of recombinant pharmaceutical proteins in plants[J].Nat Rev Genet.2003,4(10):794-805.
[13] Drake PM,Chargfelegue D,Vine ND,et al.Transgenic plants expressing antibodies:a model for phytoremediation[J].FASEB J.2002,16(14):1855-1860.
[14] Ma JK,Vine ND.Plant expression systems for the production of vaccines[J].Curr Top Microbiol Immunol.1999,236:275-292.
[15]杨振泉,刘巧泉,焦新安.利用转基因植物表达药用蛋白[J].中国生物工程杂志,2004,24(3):22-25.
[16]江小玲,贺竹梅,俞守义等.转基因植物口服疫苗的安全性问题所引发的思考[J].医学与社会,2004,17(2):17-18.
[17]苏钢.转基因蔬菜口服疫苗研究进展[J].邢台师范高专学报,2002,17(2):65-67.
[18]马三梅,王永飞,王亚琴.利用转基因植物生产可食疫苗[J].生命的化学,2004,24(1):22-24.
[19]刘红莉,雷霆,王一理.转基因植物疫苗的研究策略[J].中国生物工程杂志,2004,24(4):30-33.
[20] Yu J,Langridge W H.A plant-based multicomponent vaccine protects mice from enteric diseases[J].Nat Biotechnol,2001,19:548-552.
[21] Herman EM,Melroy DL,Buckhout TJ(1990) Apparent processing of a soybean oil bodymembrane protein accompanies the onset of oil mobilization.Plant Physiol 94: 341-349.
[22] Ma J K,Hiatt A,Hein M,et al.Generation and assembly of secretory antibodies in plants[J].Science,1995,286(5211):716-719.
[23]耿德贵,王义琴,李文彬等.利用转基因植物生产口服疫苗的研究现状[J].生物工程进展,2002,22(1):19-210.
[24]李轶女,胡英考.转基因植物基因工程疫苗[J].生物技术通报,2002,2:11-15.
[25]凌华,黄惠琴,鲍时翔.植物生物反应器研究进展[J].中国生物工程杂志,2002,22(5):21-25.
[26]任志强,刘惠民,李润植等.转基因植物作为生物反应器在疫苗生产中的应用[J].中国生物工程杂志,2003,23(6):31-35.
[27]王瑞刚,王水平.外源蛋白表达系统及利用植物表达外源蛋白的特点与优势[J].生物学通报,2003,38(1):11-12.
[28] Giddings G,Allison G,Brooks D,et al,Transgenic plants as factories for biopharmaceutical[J].Nat Biotechno1,2000(18):1151-1155.
[29] Twyman RM,Stoger E,Schillberg S,Christou P,Fischer R (2003) Molecular farming in plants: host systems and expression technology.Trends Biotechnol 21:570-578.
[30] Haq T A,Mason H S,Clements J D,et al.Oral immunization with recombinant bacterial antigen produced in transgenic plants.Science,1995,268:714-716.
[31] Mason H S,Haq T A,Clements J D,et al.Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin(LT):potatoes expressing a synthetic LT-B gene.Vaccine,1998, 16:1336-1343.
[32] Streatfield S J,Jilka J M,Hood E E,et al.Plant-based vaccines:unique advantages.Vaccine, 2001,19:2742-2748.
[33] Arakawa T,Chong D K X,Langridge W H R.Efficacy of a food plant based oral cholera toxin B subunit vaccine.Nature Biotechnol,1998,16:292-297.
[34] Jani D,Singh N K,Bhattacharya S,et al.Studies on the immunogenic potential of plant- expressed cholera toxin B subunit.Plant Cell Rep,2004,22:471-477.
[35] Mason H S,Ball J M,Shi J J,et al.Experssion of Nowalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.Proc Natl Acad Sci USA,1996,93:5335-5340.
[36] SandhuJ S,Krasnyanski S F,Dormer L L,et al.Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-F Protein induces a systemic immune response.Transgenic Res,2000,9:127-135.
[37] Muller C P,Fack F D,Damien et al.Immunogenic measles antigens expressed in plants: role as an Edible vaccine for adults.Vaccine,2003,21:816-819.
[38] Khandelwal A,Renukaradhya G J,Rajasekhar M,et al.Systemic and oral immunogenicity of hemagglutinin protein of rinderpestvirus expressed by transgenic peanut plants in a mouse model.Virology,2004,323:284-291.
[39] Karasev A V,Foulke S,Wellens C,et al.Plant based HIV-l vaccine candidate:Tat protein produced in spinach.Vaccine,2005,23:1875-1880.
[40] Arakawa T,Yu J,Langridge W H R.Synthesis of a cholera toxin B subunit-rotavirus NSP4fusion protein in potato.Plant Cell Rep,2001,20:343-348.
[41] Kim T G,Langridge W H R.Synthesis of an HIV-1Tat transduction domain-rotavius enterotoxin Fusion protein transgenic potato.Plant Ceill Rep,2004,22:382-387.
[42] Wigdorovitz A,Mozgovoj M,Dus Santos M J et al.Protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants[J].Gen.Virol, 2004,85:1825-1832.
[43] Perez Filgueira D M,Mozgovoj M,Wigdorovitz A,et al.Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8* produced in plants using a TMV-based vector.Arch Virol,2004,149:2337-2348.
[44] Matsumura T,Itchoda N,Tsunemitsu H.Porduction of immunogenic VP6 protein of bovine group A rotavirus in transgenic potato plants.Arch Virol,2002,147:1263-1270.
[45] Yu J,Langridge W.Expression of rotavirus capsid protein VP6 in transgenic potato and its oral immunogenicity in mice.Transgenic Res,2003,12:163-169.
[46] Wu Y Z,Li J T,Mou Z R,et al.Immunization with rotavirus VP7 expressed in transgenic potatoes induced high titers of mucosal neutralizing IgA.Virology,2003,313:337-342.
[47] Bishop R.R.,Davidson G.P.,Holmes I. H.et al.Virus Particles in epithelial cells of duodena mucosa from children with acute non-bacterial gastroenteritis.Lancet.1973,2:1281-1283.
[48] ESTES M K,COHEN J.Rotavirus gene structure and function[J].Microbiol Rev,1989, 53(4) :410-419.
[49] Institute of Medicine.The prospects for immunizing against rotavirus in new vaccine development.Establishing Priorities.Diseases of importance in developing countries.VOlII Washington,DC:National Academy Press,1986,318-388.
[50]王春凤,丛彦龙.轮状病毒研究现状[J].人畜共患传染病防治研究新成果汇编.
[51] Kalica AR,Flores J,Greenberg HB.Identification of the rotaviral gene that codes for hemagglutinin and protease-enhanced plaque formation.Virol.1983,125:194-205.
[52] Davidson G. P,Goller I,Bishop R. F.et al.Immunoflunrescence in duodenal mucosa of children with acute enteritis due to a new virus[J].Clin.Pathol.1975,28:263-266.
[53] Greenberg H.G.Clack H.E.Offit P.A.Rotavirus pathology and pathophysiology.Curr.Top. Microbiol.Immunol.1994.185:255-283.
[54] Stephen. J.Functional abnormalities in intestine.In“Viruses and the Gut”,proceedings of the Ninth BSG SKSF International Workshop(M.J.G. Farthing,Ed.) PP.41-44.Swan Press. London,1989.
[55] Del Castillo J.R.,Ludert J.E,Sanchez A.et al.Rotavirus in fection alters Na+ and K+ homeostasis in MA-104 cells[J].Gen.Virol.1991,72:541-547.
[56] Michelangeli F.Ruiz M.C.Del Castillo J.R.,et al.Effect of rotavirus infection on Intracellular calcium homeostasis in cultured cells.Virology,1991,181:520-527.
[57] Tian P.Ester M.K.,HuY.et al.The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca from the endoplasmic reticulum[J].Virol.1995,69:5763-5772.
[58] Ward L.A.,Rosen B.L.Yuan L.et al.Pathogenesis of an attenuated and a virulent strain of group A human rotavirus in neonatal gnotobiotic pigs[J].Gen.Virol,1996,77:1431-1441.
[59] Eglee Perez M,Glass R,Alvarez G,Pericchi LR,Gonzalez R,et al.(2000) Rhesusrotavirus-based quadrivalent vaccine is efficacious despite age,socioeconomic conditions and seasonality in Venezuela.Vaccine 19:976-981.
[60] Estes MK,Conner ME,Gilger MA,Graham DY(1989) Molecular biology and immunology of rotavirus infections.Immunol Invest 18:571-581.
[61] Affranchino J.L.,Pollevick,G.and Frasch,A.C.C.(1991)The expression of the major shedTrypanosoma cruzi antigen results from the developmentally-regulated transcription of a small gene family.FEBS Lett 2:316-320.
[62] McNeal MM,VanCott JL,Choi AH,Basu M,Flint JA,et al.(2002) CD4 T cells are the only lymphocytes needed to protect mice against rotavirus shedding after intranasal immunization with a chimeric VP6 protein and the adjuvant LT(R192G)[J].Virol 76:560-568.
[63] Choi NW,Estes MK,Langridge WH (2005) Oral immunization with a shiga toxin B subunit: rotavirus NSP4(90) fusion protein protects mice against gastroenteritis.Vaccine 23: 5168-5176.
[64] Herrmann JE,Chen SC,Fynan EF,Santoro JC,Greenberg HB,et al.(1996) Protection against rotavirus infections by DNA vaccination. J Infect Dis 174 Suppl 1:93-97.
[65] Steele AD,Basetse HR,Blacklow NR,Herrmann JE(1998) Astrovirus infection in South Africa: a pilot study.Ann Trop Paediatr 18:315-319.
[66] Xu WC,Chen BT,Fu JX(1997) [Progress in the study on the treatment of rotaviral enteritis with traditional Chinese medicine].Zhongguo Zhong Xi Yi Jie He Za Zhi 17:511-512.
[67] Chen HN,Dennehy PH,Oh W,Lee CN,Huang ML,et al.(1997) Outbreak and control of a rotaviral infection in a nursery[J].Formos Med Assoc 96:884-889.
[68] Chen SC,Fynan EF,Robinson HL,Lu S,Greenberg HB,et al.(1997) Protective immunity induced by rotavirus DNA vaccines.Vaccine 15:899-902.
[69] Mat sumura T,Itchoda N,Tsunemtsu H.Production of immunogenic VP6 protein of bovine group A rotavirus in transgenic potato plants[J].Arch Virol,2002,147:1263-1270.
[70] Hardegger M,Sturm A.Transformation and regeneration of carrot (Daucus Carota L.)[J].Molecular Breeding,1998,4:119-127.