乙肝病毒表面抗原（HBsAg）、戊肝病毒ORF2基因片段NE_2在番茄中的表达及免疫活性
|
摘要
本研究以番茄子叶和下胚轴为材料，以Gus瞬时表达效率为指标，系统研究了农杆菌介导的番茄遗传转化的影响因素，在此基础上建立并优化了番茄“羞女”品种的遗传转化体系；构建乙型肝炎病毒表面抗原基因(HBsAg)、戊型肝炎病毒ORF_2基因片段NE_2(HEV-NE_2)的植物表达载体，利用已建立的转化体系将其导入番茄，获得了经PCR和Southern杂交证实的转基因植株。ELISA检测结果显示在番茄中表达的目的蛋白具有较好的免疫活性；动物试验结果初步表明，用转基因番茄表达的HBsAg口服或肌注初免过的小鼠，均有不同程度的加强免疫效果。展示了用番茄研制乙肝、戊肝口服疫苗的美好前景，为推动转基因植物口服疫苗走向实用化提供实验基础和理论指导。
1．植物表达载体构建：利用含GUS和去GUS的中间载体pCAMBIA1301和pCAMBIA1300构建了乙肝病毒表面抗原(HBsAg)、戊肝病毒ORF_2基因片段NE_2的植物表达载体p1301HBs、p1300HBs、p1301NE_2和p1300NE_2。其中含CaMV35s启动子、nos终止子、Ω增强子、Kan抗性筛选标记Kan~r和潮霉素磷酸转移酶基因(HPT)。经酶切鉴定后直接法导入农杆菌菌株LBA4404、EHA105、AGL1。
2．农杆菌介导的番茄遗传转化体系的建立和优化：以含质粒p1301HBs(含Gus基因)的根癌农杆菌侵染番茄外植体，以Gus瞬时表达效率为指标，系统研究了农杆菌菌株、外植体年龄、预培养时间、共培养时间、培养条件、筛选条件等对转化效率的影响并在此基础上建立、优化了番茄“羞女”品种的遗传转化体系。
福建农林大学博士学位论文 中文摘要
即：EHA 105菌株；外植体苗龄gd暗光下预培养3d，或苗龄12d
暗光下预培养Zd：农杆菌侵染后暗光下共培养3小筛选剂潮霉素
（Hrr）浓度为20mm／L，杀菌剂前期用梭芋青霉素（Carb），后期
用头抱霉素（Cef），浓度初期 500mg／L，以后逐渐降低。
将该体系运用于去 GUS标记的 PI 300HBS和戊肝病毒基因的
转化，得到了较好的结果，说明该体系是可靠、可重复、有效的。
3．转基因苗的鉴定及目的蛋白的定量：抗性苗总DNA经
PCR、Southern杂交证实目的基因己整合到番茄基因组中，ELISA
检测结果证明在番茄中正确表达了具有免疫活性的乙肝病毒表面
抗原蛋白门BSAg人戊肝病毒ORFZ基因片段NEZ（HEV－NEZ人
根据ELISA定量结果，HBSAg在番茄中最大表达量约为240刁00
us／s，约占总可溶性蛋白的0．003％，而eV－NEZ的表达量较低。
目前己扩繁转HBSAg的转基因苗40多株，已结番茄100多个，保
存有转HEV十 的转基因苗7株，其中有一株己结两个果实。
4．小鼠免疫试验：以0．sllg／g亚免疫剂量初兔的小鼠，4
周后用转HBSAg的转基因组织饲喂，至第8周抗体由饲喂前的
0．4上升到互．0以上；用浓缩、纯化过的植物蛋白加强免疫初免
过的小鼠，也发现有较强的特异性抗体上升。说明用番茄表达的
重组HBSAg具有免疫原性。
By using cotyledons and hypocotyls of tomatos as transformatic explants, the factors affecting gene transfer mediated by Agrobacterium tumefaciens were systematically examined according to Gus expressing efficiency, and the optimized genetic transformation system of tomato was established. The plant binary expression vector p!301HBs, pBOOHBs carrying hepatitis B virus surface antigen(HBsAg) gene, p!301NE2, p!300NE2 carrying NE2 gene, a fragment of ORF2 region of hepatitis E virus (HEV-NE2), were constructed and introduced into tomato, and hygromycin-resistant plantlets were obtained. The results of PCR and Southern blotting of tomato genomic DNA demonstrated that the target gene was integrated into the genome of tomato plants. The examination of ELISA suggested the HBsAg gene has expressed correctly in transgenic tomato plants. The immunization experiment indicated that the HBsAg-transgenic plant derived vaccine can produce booster immune responses in Balb/c mice primed with commercial vaccine. All these demonstrated the perfect perspective of plant-derived edible vaccine and provided experimental basis and guiding theory for the practicality of plant-derived edible vaccine.
1.Construction of plant binary expression vector: The HBsAg gene linked with CaMV35S promoter, nos terminal and Q enhancer was inserted into the mediate expression vectors pCAMBIA1301 (containing hygromycin- resistant gene, kanamycin-resistant gene, Gus gene) and pCAMB!A1300 (containing Hyg- resistant gene, Kan-resistant gene, no Gus gene), respectively, to form the reconstructed plant binary expression plasmids p!301HBs and p!300HBs. In the same way, the plant binary expression plasmids p!301NE2, p!300NE2 carrying HEV-NE2 gene were constructed. Restriction endonuclease analysis indicated that these plasmids were directly introduced into strains LBA4404, EHA105, AGL1.
2. Genetic transformation system establishment: Genetic transformation conditions were examined by Gus expression, and an efficient genetic transformation system was developed. That is: 9-days-old explants pre-cultured for 3 days in darkness or 12-days-old explants pre-cultured for 2 days in darkness were co-cultured with EH A105 for 3 days in darkness, then selected by 20mg/g hygromycin. 500mg/g carbenicillin(carb) which will be replaced by cefotaxine(cef) later was used as inhibitor of the Agrobacterium tumefaciens, both of which will be reduced gradually.
Using the above-mentioned transformation system, the p!300HBs without Gus gene, and the HEV-NE2 gene were introduced into tomato, many hyg-resistent plantlets were acquired too. This demonstrated that the established transformation system is reliable, repeatable, thus efficient.
3. Distinguishing the hyg-resistant plantlets and determining expression amount of target protein: The integration of foreign DNA into transgenic tomato was confirmed by hygromycin resistance, Gus assay, PCR and Southern blotting. The immunoacticity of recombinant HBsAg, HEV-NE2 was shown by ELISA. The amount of HBsAg in transgenic tomato is about 240-300ng/g fresh weight, and 0.003 percent of total soluble protein, whereas the amount of HEV-NE2 is very low. Up to now, we have obtained about 40 HBsAg-transgenic plants bearing more than 100 tomato fruits, 7 NE2~transgenic plants, one of which has born 2 fruits.
4. Immunogenicity of transgenic plant-derived HBsAg: Mice primed with injection of a sub-immunogenic dose(0.5 u g/g) of commercial vaccine, were fed with transgenic tissue or injected with purified extraction. Four weeks later, the mice boosted special serum antibody of HBsAg.
1．植物表达载体构建：利用含GUS和去GUS的中间载体pCAMBIA1301和pCAMBIA1300构建了乙肝病毒表面抗原(HBsAg)、戊肝病毒ORF_2基因片段NE_2的植物表达载体p1301HBs、p1300HBs、p1301NE_2和p1300NE_2。其中含CaMV35s启动子、nos终止子、Ω增强子、Kan抗性筛选标记Kan~r和潮霉素磷酸转移酶基因(HPT)。经酶切鉴定后直接法导入农杆菌菌株LBA4404、EHA105、AGL1。
2．农杆菌介导的番茄遗传转化体系的建立和优化：以含质粒p1301HBs(含Gus基因)的根癌农杆菌侵染番茄外植体，以Gus瞬时表达效率为指标，系统研究了农杆菌菌株、外植体年龄、预培养时间、共培养时间、培养条件、筛选条件等对转化效率的影响并在此基础上建立、优化了番茄“羞女”品种的遗传转化体系。
福建农林大学博士学位论文 中文摘要
即：EHA 105菌株；外植体苗龄gd暗光下预培养3d，或苗龄12d
暗光下预培养Zd：农杆菌侵染后暗光下共培养3小筛选剂潮霉素
（Hrr）浓度为20mm／L，杀菌剂前期用梭芋青霉素（Carb），后期
用头抱霉素（Cef），浓度初期 500mg／L，以后逐渐降低。
将该体系运用于去 GUS标记的 PI 300HBS和戊肝病毒基因的
转化，得到了较好的结果，说明该体系是可靠、可重复、有效的。
3．转基因苗的鉴定及目的蛋白的定量：抗性苗总DNA经
PCR、Southern杂交证实目的基因己整合到番茄基因组中，ELISA
检测结果证明在番茄中正确表达了具有免疫活性的乙肝病毒表面
抗原蛋白门BSAg人戊肝病毒ORFZ基因片段NEZ（HEV－NEZ人
根据ELISA定量结果，HBSAg在番茄中最大表达量约为240刁00
us／s，约占总可溶性蛋白的0．003％，而eV－NEZ的表达量较低。
目前己扩繁转HBSAg的转基因苗40多株，已结番茄100多个，保
存有转HEV十 的转基因苗7株，其中有一株己结两个果实。
4．小鼠免疫试验：以0．sllg／g亚免疫剂量初兔的小鼠，4
周后用转HBSAg的转基因组织饲喂，至第8周抗体由饲喂前的
0．4上升到互．0以上；用浓缩、纯化过的植物蛋白加强免疫初免
过的小鼠，也发现有较强的特异性抗体上升。说明用番茄表达的
重组HBSAg具有免疫原性。
By using cotyledons and hypocotyls of tomatos as transformatic explants, the factors affecting gene transfer mediated by Agrobacterium tumefaciens were systematically examined according to Gus expressing efficiency, and the optimized genetic transformation system of tomato was established. The plant binary expression vector p!301HBs, pBOOHBs carrying hepatitis B virus surface antigen(HBsAg) gene, p!301NE2, p!300NE2 carrying NE2 gene, a fragment of ORF2 region of hepatitis E virus (HEV-NE2), were constructed and introduced into tomato, and hygromycin-resistant plantlets were obtained. The results of PCR and Southern blotting of tomato genomic DNA demonstrated that the target gene was integrated into the genome of tomato plants. The examination of ELISA suggested the HBsAg gene has expressed correctly in transgenic tomato plants. The immunization experiment indicated that the HBsAg-transgenic plant derived vaccine can produce booster immune responses in Balb/c mice primed with commercial vaccine. All these demonstrated the perfect perspective of plant-derived edible vaccine and provided experimental basis and guiding theory for the practicality of plant-derived edible vaccine.
1.Construction of plant binary expression vector: The HBsAg gene linked with CaMV35S promoter, nos terminal and Q enhancer was inserted into the mediate expression vectors pCAMBIA1301 (containing hygromycin- resistant gene, kanamycin-resistant gene, Gus gene) and pCAMB!A1300 (containing Hyg- resistant gene, Kan-resistant gene, no Gus gene), respectively, to form the reconstructed plant binary expression plasmids p!301HBs and p!300HBs. In the same way, the plant binary expression plasmids p!301NE2, p!300NE2 carrying HEV-NE2 gene were constructed. Restriction endonuclease analysis indicated that these plasmids were directly introduced into strains LBA4404, EHA105, AGL1.
2. Genetic transformation system establishment: Genetic transformation conditions were examined by Gus expression, and an efficient genetic transformation system was developed. That is: 9-days-old explants pre-cultured for 3 days in darkness or 12-days-old explants pre-cultured for 2 days in darkness were co-cultured with EH A105 for 3 days in darkness, then selected by 20mg/g hygromycin. 500mg/g carbenicillin(carb) which will be replaced by cefotaxine(cef) later was used as inhibitor of the Agrobacterium tumefaciens, both of which will be reduced gradually.
Using the above-mentioned transformation system, the p!300HBs without Gus gene, and the HEV-NE2 gene were introduced into tomato, many hyg-resistent plantlets were acquired too. This demonstrated that the established transformation system is reliable, repeatable, thus efficient.
3. Distinguishing the hyg-resistant plantlets and determining expression amount of target protein: The integration of foreign DNA into transgenic tomato was confirmed by hygromycin resistance, Gus assay, PCR and Southern blotting. The immunoacticity of recombinant HBsAg, HEV-NE2 was shown by ELISA. The amount of HBsAg in transgenic tomato is about 240-300ng/g fresh weight, and 0.003 percent of total soluble protein, whereas the amount of HEV-NE2 is very low. Up to now, we have obtained about 40 HBsAg-transgenic plants bearing more than 100 tomato fruits, 7 NE2~transgenic plants, one of which has born 2 fruits.
4. Immunogenicity of transgenic plant-derived HBsAg: Mice primed with injection of a sub-immunogenic dose(0.5 u g/g) of commercial vaccine, were fed with transgenic tissue or injected with purified extraction. Four weeks later, the mice boosted special serum antibody of HBsAg.
引文
[1] 戈峰，李典模．可持续农业中的害虫管理问题．昆虫知识，1997，34(1)：39-45
[2] Fischhoff DA, Bowdish KS, Perlak FJ, et al. Insect tolerant tomato plants. Biotechnology, 1987, 5:807-813.
[3] 王关林，方宏筠主编．植物基因工程原理与技术．科学出版社，1998
[4] 曹孟良，转基因植物研究及其产业化进展．湖南农业科学，2000(4)：6-8
[5] Daniell H. Environmentally friendly approaches to genetic engineering. In viro Cell. Dev. Biol. Plant Sci. 1999,35,361-168
[6] 范云六，张春义．迎接21世纪农作物生物技术的挑战．生物技术通报，1999(5)：1-6
[7] Dessalegne L, Wetten AC, Caligari PDS. Production of transgenic tomatoes expression oxalateoxidase. Acta Hortic, 1997, 447:457-458.
[8] 付永彩，丁月云，刘新仿，等．抑制衰老的嵌合基因在水稻中的转化．科学通报，1998，43(18)：1963-1967
[9] 黄永芬，汪清胤，付桂荣．美洲拟鲽抗冻蛋白基因(afp)导入番茄的研究．生物化学杂志，1997，13(4)：418-422．
[10] 雷茂良，程金根．全球转基因植物发展现状．生物技术通报，1998(6)：30-32
[11] 贾士荣．转基因植物的环境及食品安全性．生物工程进展，1997，17(6)：36-41
[12] 黄其满．农业生物技术产业发展问题的思考．高技术通讯，2000(6)：107-109
[13] 王锡华，张峰，赵遵田．我国转基因植物研究进展．山东科学，1999(2)：32-36
[14] 王亮，游松．转基因植物的现状．沈阳药科大学学报，2001，18(3)：217-222
[15] Powell Abel P, Nelson RS, De B, et al. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science, 1986, 232:738-743
[16] Whitham S, McCormick S, Baker B.The N gene of tobacco confers resistance to tobacco mosaic virus in transgenic tomato. Proc Natl Acad Sci, 1996,93(16):8776-8781.
[17] 程英豪，吴光，王继伟，等．表达黄瓜花叶病毒外壳蛋白的转基因番茄抗黄瓜花叶病毒浸染．植物学报，1997，39(1)：16-31．
[18] Kunik J, Gafni Y, Citovsky W. Transgenic tomato plants expressing TYLCV capid protein a reresistant to the virus. The role of the nuclear localization signal(NLS) in the resistance. Acta Hortic, 1997, 447:387-391.
[19] Ultzen T, Gielen J, Venenca F, et al. Resistance to tomato spotted with virus in transgenic tomato hybrids. Euphtica, 1995, 85(1-3): 159-168.
[20] 赵淑珍，王昕，王革娇，等．由卫星互补DNA单体和双体基因构建的抗黄瓜花叶病毒的转基因番茄．中国科学(B辑)，1990，(7)：706-713．
[21] Thomzik JZ, Stenzel K, Stocker R. Synthesis of agrapevinephy to a lexinin transgenic tomatos (Lycopersicones-culentum Mill.) conditions resistance against phytophtho rain festans. Physiol Mol Plant Pathol, 1997, 51(4): 265-270.
[22] Jongedij kE, Tiglaar H, Roekel JSC, et al. Synergistic activity of chitimases and β-1,3-glucanases enhances fun-gal resistance in transgenic tomato plants. Euphytica, 1995, 85(1-3): 173-180.
[23] Riely BK, Martin GB. Ancient origin of pathogen recognition specificity conferred by the tomato disease resistance gene Pto. PNAS, 2001, 98: 2059-2064
[24] Vander ST, Bosch D, Hones G, et al. Insect resistance of transgenic plants that express modified Bacillus thuringiensis CryIA(b) and CryIcC genes; a resistance management strategy. Plant Mol Biol, 1994, 26(1):51-59.
[25] Janses S, De Clercq R, Reynaerts A, et al. Green house evaluation of transgenic tomato plants expressing a Bacillus tharingiensis insecticidal crystal protein, for control of Helicoverpaarmigera (Lepidoptera Noctuidae). Meded Fac Land houwwet Univ Gent, 1992, 57(2b): 515-522.
[26] Rhim S-L, Cho HJ. Development of insect resistance in tomato plants expression the δ-endo toxin gene of Bacillus thuringiensis Subsptene brionis. Mol Breed, 1995,1(3): 229-236.
[27] Williamson VM. Root-knot nematode resistance genes in tomato and their potential for future use. Annu. Rev. Phytopathol., 1998,36:277-293
[28] DeBlock M, Botterman J, Vandewiele M, et al. Engineering herbicide rsistance in plants by expression of a detoxifying enzyme. EMBOJ, 1987,6(9):2513-2518.
[29] Benbrook C. Hrbicide resistance: environmental and economic tissues. Proc Bio Expo Butterworth Boston, 1986,(86):27-34.
[30] Fillatti JJ, Haissig B, Mccown B, et al. Development of glyphosate tolerant populus plants through expression of amutant aroagene from salmonellaty phimurium. Basic Life Sci, 1988,44:243-249.
[31] Smith CJS. Inheritance and effect on ripening of antisense polygalacturonase gene in transgenic tomatos. Plant Mol Biol, 1990, 14:369～379
[32] 叶志彪，李汉霞，周国林．番茄多聚半乳糖醛酸反义cDNA克隆的遗传转化与转基因植株再生．园艺学报，1994，21(3)：305-308．
[33] Tucker G, Zhang J. Expression of polygalacturonase and pect inesterase in normal and transgenic tomatoes. Prog Biotechnol, 1996,14:347-354
[34] Oller PW, Min-wong L, Taylor LP, et al. Reversible inhibition of tomato during fruit senescence by antisense RNA. Science, 1991,254:437-439.
[35] 仇润祥，扈延茂，王永胜，等．番茄1-氨基环丙烷羧酸(ACC)合成酶反义RNA-核酶嵌合DNA序列的构建．遗传，1999，1：1-6．
[36] 叶志彪，李汉霞，刘勋甲．利用转基因技术育成耐贮藏番茄——华番1号．中国蔬菜，1999，(1)：6-10．
[37] Klee HJ, Hayford MB, Kretzmer KA, et al. Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants. Plant Cell, 1991,3(11):1187-1193.
[38] Kramer MG, Kellogy J, Wagoner W, et al. Reduced ethylene synthesis and ripening control in tomatoes expressing sad enosy Imethionine hydrolase. NATOASISer, 1997, 34:307-319.
[39] Hightower R, Baden C, Penzes E, et al. Expression of antifreeze proteins in transgenic plants. Plant Mol Biol, 1991,17(5):1013-1021
[40] Penarrubia Loia, Rosalindkim, James Giovannoni. Production of the sweet protein monellin in transgenic plants. Biotechnology, 1994(10): 561-564
[41] Klann M, Hall B, Bennett AB. Antisense acid invertase(T1V1) gene alters soluable sugar composition and size in tomato fruit. Plant Physiol, 1996, 112(3):1321-1330.
[42] Drake CR, Bird CR, Schuch WW. Over expression of phytoenesynthase transgene in tomato plants for enhanced carotenoidcontent. PcT Int Appl WO 9746,690(CI,C12N15/67), 1997-12-11. GBAppl,96/11,981, 1996-06-07, 3.
[43] Bramley PM. The regulation and genetic manipulation of carotenoid
biosynthesis in tomato fruit. Pure Appl Chem, 1997, 69(10) : 2159-2162．
[44] Martineau B, Adams DF, Deverna JW. Ovaryprr dominant expression of acytokin in biosynthetic gene in tomato results in highe totaland soluable fruit solids .Proc Plant Growth Regul Soc Am, 1 994,2 1 : 1 5-24．
[45] Tieman DM, Kausch KD, Serra DM. et al. Field performance oftransgenic tomato with reduced pectin methylesterase activity.Jam Soc Hortic Sci,1995,120(5) :765-768
[46] Mariani C. A chimaeric ribonuclease-inhibitor gene restors fertility to male sterile plants. Nature, 1992,357: 384-387
[47] 张宏，王波．雄性不育嵌合基因的构建及番茄转化研究．遗传，1998,20 (3) : 5-7
[48] Paul, Woeller, LuMin-Wong. Reversible inhibition of tomato of ruit senescence by antisense RNA. Science, 199 1,254:437-439
[49] Bird CR. Using antisense RNA to study gene function inhibition of caroteniod biosynthesis in transgenic tomatoes. Biotechnology, 1991(9) : 635-639
[50] JohnI, DrakeR, FurrellA. Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants molecular and physiological analysis. Plant, 1995, 7(3) :483-490．
[51] 贺竹梅，曹俊，李宝健，等．人促红细胞生成素基因在番茄中的表达．遗传学报,1998, 25 (2) : 155-159
[52] Daniell H. GM crops: public perception and scientific solutions. Trends Pkant Sci. 1999,4,467-469
[53] Matsumoto S, Ikuar K, Ueda M. Characterization of a human glycoprotein (erythropoietin, EPO) produced in cultured tobacco cells, Plant Molecular Biology, 1995,27: 11 63
[54] Mori M, Zhang GH, Kaido M, et al. Efficient production of human gammainterferon in tobacco protoplasts by genetic ally engineer edbrome mosaic virus RNAS. Journal of General Virology,1993,74:1225
[55] Zeitlin L, Olmsted SS, Moench, et al. A humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genitalherpes . Nature Biotechnology, 1998,16:1361
[56] Mason HS, Lam DMK, Arntzen CJ, et al. Expression of hepatitis Bsurface antigen in transgenic plants. Proc. Natl.Acad. Sci. 1992, 89, 1 1745-1 1749
[57] ThanavalaY, Yang YF, Lyons P, et al. Immunogenicity of transgenic plant-derived hepatitisB surface antigen. Proc. Natl. Acad. Sci. 1995,
92,3358-3361
[58] Richter LJ, Thanavala Y, Arntzen CJ, et al. Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat. Biotechnol. 2001,18, 1167-1171
[59] Kapusta J, Modelska A, Figlerowicz M, et al. A plant-derived edible vaccine against hepatitis B virus. FASEB J.1999 ,13, 1796-1799
[60] 刘德虎．利用转基因植物生产药用蛋白．生物技术通报，1999,(4) :1-5
[61] 赵春晖，王荣，赵长生，等．含与不含前导序列的乙肝病毒表面抗原在转基因番茄中表达的研究．农业生物技术学报，2000, 8(2) : 190-193
[62] Haq TA , Mason HS, Clements JD, et al. Oral immunization with arecombinant bacterial antigen produced intransgenic plants. Science, 1995,268,714-716
[63] Lauterslager TGM, Florack DEA, van der Wal TJ, et al.Oral immunisation of naive and primed animals with transgenic potato tubers expressing LT-B. Vaccine, 2001 19,2749-2755
[64] Mason HS, Haq TA, Clements JD 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
[65] Tacket CO, Mason HS, Losonsky G, et al. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat. Med. 1998,4,607-609
[66] Curtiss RI, Cardineau CA. Oral immunisation by transgenetic plants. World Patent Application , WO 1990,90/02484
[67] Tacket CO, Reid RH, Boedeker EC, et al. Enternal immunisation and challenge of volunteers given enterotoxigenic E. coli CFA/II encapsulated in biodegradable microspheres. Vaccine, 1994,12,1270-1274
[68] Streatfield SJ, Jilka JM, Hood EE, et al. Plant-based vaccines: unique advantages. Vaccine, 2001,19, 2742-2748
[69] Arakawa T, Chong DKX, Merritt JL, et al. Expression of choleratoxin B subunit oligomers in transgenic potatoplants. Transgenic Res. 1997, 6, 403-413
[70] Arakawa T, Chong DKX, Langridge WHR, et al. Efficacy of a food plant-basedoral cholera toxin B subunit vaccine. Nat.Biotechnol. 1998,16, 292-297
[71] Mason HS, Ball JM, Shi J-J, Jiang X,et al. Expression of Norwalk virus
capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc. Natl. Acad. Sci. 1996, 93, 5335-5340
[72] Jacket CO, Mason HS, Losonsky G, et al. Human immune responses to a novel Norwalk virus vaccine delivered in transgenic potatoes. J. Infect. Dis. 2000, 182,302-305
[73] McGarvey PB, Hammond J, Dienelt MM, et al. Expression of therabies virus glycoprotein in transgenic tomatoes. Biotechnology, 1995, 13, 1484-1487
[74] YusibvoV, Modelska A, Steplewski K, et al. Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1． Proc. Natl.Acad. Sci. 1997,94,5784-5788
[75] Modelska A, Dietzschold B, Sleysh N, et al. Immunization against rabies with a plant-derived antigen. Proc. Natl.Acad. Sci. 1998,95,2481-2485
[76] SugiyamaY, Hamamoto H, Takemoto S, et al. Systemic production of foreign peptides on the particles surface of tobacco mosaic virus. FEBS Letters, 1995, 359(2-3) ,247-250
[77] Huang Z, Dry I, Webster D, et al. Plant-derived measles virus hemagglutin in protein induces neutralizing antibodies in mice. Vaccine, 2001,19,2163-2171
[78] Tackaberry ES, Dudani AK, Prior F, et al. Development of biopharmaceuticals in plant expression systems: cloning, expression and immunological reactivity of human cytomegalovirus glycoprotein B (UL55) in seeds of transgenic tobacco. Vaccine, 1999, 17, 3020-3029
[79] Turpen TH, Rein! SJ, Charoenvit Y, et al. Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Biotechnology, 1995,13,53-57
[80] Carrillo C, Wigdorovitz A, Oliveros JC, et al. Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants. Journal of General Virology, 1998, .72, 1688-1690
[81] Wigdorovitz A, Carrillo C, Dus Santos MJ, et al. Induction of a protective antibody response to foot and mouth disease in mice following oral or parenteral immunization with alfalfa transgenic plantsexpressing the viral structural protein VP1． Journal of General Virology , 1999 ,255, 347-353
[82] Porta C, Spall VE, Loveland J, et al. Development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides. Journal of General Virology, 1994,202,949-955
[83] Castanon S, Marin MS, Martin-Alonso JM. et al. Immunization with
potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus. Journal of General Virology, 1999,73, 4452-4455
[84] Gomez N, Caxrillo C, Salinas J, et al. Expression of immunogenic glycoprotein S polypeptides from transmissible gastroenteritis coronavirus in transgenic plants. Journal of General Virology, 1998,249, 352-358
[85] Tuboly T, Yu W, Bailey A, et al. Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants. Vaccine, 2000, 18, 2023-2028
[86] Dalsgaard K, Urtenthal A, Jones TD, et al. Plant-derived vaccine protects target animals against a viral disease.Nat. Biotechnol. 1997,15,248-252
[87] Fernandez MR, Martinez-Torrecuadrada JL. Development of an antigen presentation system based on plum pox potyvirus . FEBS Letters, 1998, 27,229-235
[88] 蒋少云，凌均启．转基因番茄防龋疫苗的初步研究．口腔医学纵横杂志，2000, 16 (2) , 94-96
[89] Maygd LS, Panis B, Remy S, et al, Generation of transgenic banana (musa acuminata) plants via agrobacterium-inediated transformation. Biotechnology, 1995, 13, 486-492
[90] Moffat AS. Exploring Transgenic plants as a new vaccine source. Science, 1995, 268(5211 ):658-660
[91] Staub JM, Garcia B, Graves J, et al. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 2000, 18(3) , 333-338
[92] Kim J, Mayfield PS. Protein disulfide isomerase as a regulator of chloroplast translational activation. Science, 1997,278,1954-1957
[93] Reulland E, Miginiac-Maslow M. Regulation of chloroplast enzyme activities by thioredoxins:activation or relief from inhibition. Trends Plant Sci. 1999,4,136-141
[94] De Cosa B, Moar W,Lee SB,et al. Hyper-expression of the Bt Cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat. Biotechnol. 2001, 19(1 ),71-74
[95] Mclnerney TL. Analysis of the ability of five adjuvants to enhance immune responses to a chimeric plant virus displaying an HIV-1 peptide. Vaccine, 1999, 17:11-12,1359-1368
[96] Qingxian Kong, Liz Richter, Yu Fang Yang, et al. Oral immunization with hepatitis B surface antigen expressed in transgenic plants. PNAS, 2001,
98(20) : 11539-11544
[97] Brennan FR. Pseudomonas aeruginosa outer-membrane protein F epitopes are highly immunogenic in mice when expressed on a plant virus. Microbiology, 1999,145:211-220
[98] Henry D, Stephen JS, Keith W. Medical molecular farming:production of antibodies,biopharmaceuticals and edible vaccines in plants. Trends in Plants Science, 2001,6(5) :219-226
[99] Liz JR, Yasmin T, Charles J A. et al. Production of HBsAg in transgenic plants for oral immunization. Nature Biotechnology, 2000,18:1167-1171
[100] Koprowski H, Yusibov V. The green revolution: plants as heterologous expression vectors. Vaccine, 2001,19:2735-2741
[101] Balayan MS, Andjaparidze AG, Savinskaya SS, et al.Evidence for a virus in non-A,non-B hepatitis transmitted via the fecal-oral route. Intervirology, 1983,20(1) :23-31． 3
[102] Reyes GR, Purdy MA, Kim JP, et al. Isolation of cDNA from the virus responsible for enterically transmittednon A, non B hepatitis. Science, 1990,247:1335-1339
[103] Purcell. In; Fields BN, Knipe DM, Howley PM, Chanock RM, Melnick JL, Monooth TP, Roizman B, Strous SE, editors. Fields Virology , 3rd ed, Philadephia: Lippincott-Raven, 1996, 2831-2843
[104] Amanda M Walmsley, Charles J Arntzen. Plants for delivery of edible vaccines. Current Opinion in Biotechnology, 2000,11:126-129
[105] Tsafrir S M, Miguel A G,Kenneth E P. Perspective: edible vaccine-a concept coming of age. Trends in Microbiology, 1998,6(11) :449-453
[106] Mason HS, Tacket CO, Richter LJ, et al. Subunit vaccines produced and delivered in transgenic plants as "edible vaccines". Res. Immunol. 1998,149,71-74
[107] Purdy MA, McCaustland KA, Krawczynski K, et al. Expression of HEV trep E fusion protein containing epitopes recognized by antibodies in sera from human cases and experimentally infected primates. Arch Virol,1992,123:335-349
[108] Wong DC, Purcell RH, Sreenivasan MA, et al.Epidemic and endemic hepatitis in India: evidence for non-A/non-B hepatitis virus etiology. Lancet ,1980,2,876-878
[109] Purdy MA, Karen A, McCaustland, et al. Preliminary evidence that a trp E HEV fusion protein protects cynomolgusmacaques against challenge
with wild type HEV. J Med Virol, 1993, 41:90-94
[110] Li F, Torresi J, Locarnini SA, et al. Amino terminal epitopes are exposed when full length open reading fram 2 of hepatitis E virus is expression in Escherichiacoli, but carbory terminal epitopes are masked. J Med Virol, 1997,52:289-300
[111] Tsarev SA, Tsareva T, Emerson SU, et al. Infeativity titration of prototypestain of hepatitis virus incynomoluss monkeys. J Med Virol, 1994,43:135-147.
[112] Lu FM, Zhuang H, Zhu YH, et al. April iminary study on immune response to hepatitis E virus DNA vaccine in mice. Chinese Med J, 1996,109:919-921
[113] He J, Hoffman SL, Hayes CG, et al. DNA inoculation with a plasmid vector carrying the hepatitis E virus structral protein gene induce immunere sponce in mice. Vaccine, 1997,15(4):357-362
[114] Jameel S, Zafrullah M, Ozdener MH, et al. Expression in animal cells and charactrization of the hepatitis E virus structrul proteins. J Virol, 1996, 70:207-216
[115] 佟玉品，毕胜利，张明程，鲁健，詹美云．戊型肝炎病毒结构区基因ORF2在Pichiapastoris中的分泌表达．中华实验和临床病毒学杂志，2000，14(4)
[116] 灵晓．我国研制成功戊型肝炎疫苗．中华医学杂志，2001，81(2)
[117] 徐道东，赵章忠，王统正等主编．茄果类蔬菜栽培技术，上海科学技术出版社，1999，2
[118] [美]J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯著，金冬雁，黎孟枫等译．分子克隆实验指南，科学出版社，1999，10
[119] 贺竹梅，张绍松．番茄转基因受体系统的研究．应用与环境生物学报，1998，4(3)：247-250
[120] 叶志彪，李汉霞，周国林．番茄子叶离体培养与再生植株．华中农业大学学报，1991，13(3)：291-295
[121] 梁小友，米景九，朱玉贤等．双抗(抗病毒及抗虫)植物表达载体构建及番茄的转化鉴定．植物学报，1994，36(11)：849-854
[122] 王忠华，夏英武．转基因植物中报告基因GUS的表达极其安全性评价．生命科学，2000，12(5)
[123] 马英，张军，林顺权，夏宁邵．利用转基因植物生产基因工程疫苗．厦门大学学报(自然科学版)，2001，40(增刊2)：71—77
[124] 林顺权，宋刚，马英，曾黎辉，吕柳新．果树转基因研究进展．园艺学报，2001，28(增刊)：589-596
[125] Kyle JW, Galvin N, Vogler C, et al. β-glucuronidase assay in animal tissue[A]. In: Gallagher, S Red. Gus Protocols: Using the Gus gene as a reporter gene expression[C]. San Diego: Academic Press, 1992:189-203
[126] Peerbolte R, Leenhouts K, Hooykaas-van Slogteren GMS, et al. Clone from a shooty tobacco crown gall tumor Ⅱ: irregular T-DNA structures and organization T-DNA, methylation and conditional expression of opine genes. Plant Mol Bio, 1986, 7: 285-299.
[127] Finnegan J, McElory D. Transgene inactivation: plants fight back. Biotechnology, 1994, 12:883-888
[128] Stam M, Mof JNM, Kooter JM. The sciences of genes in transgenic plants. Annals of Botany, 1997, 79(1): 3-12
[129] Assaad FF, Tucker KL, Signer ER. Epigenetic repeat-induced gene sciencing (RIGS) in Arabidopsis. Plant Mol Bio, 1993, 22: 1067-1085.
[130] Dorer DR, Henlkoff S. Transgene repeat arrays interact with distant heterochromatin and cause scilencing in cis and trans. Genetics, 1997, 147(3): 1181-1190
[131] Albert H, Dale EC, Lee E, et al. Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J, 1995, 7(4): 649-659
[132] Vergunst AC, Hooykass PJ. Cre/lox-mediated site specific integration of Agrobacterium T-DNA in Arabidopsis thalians by transient expression of cre. Plant Mol Biol, 1998, 38:393-406
[133] Finnegan J, McElroy D. Transgene inactivation: plant fight back, Biotechnology, 1994, 12(9): 883-888
[134] 倪健刚，王立君，应成波，等．转基因番茄快速繁殖若干问题初探．宁波农业科技，1988(3)：2-5
[135] 马英，林顺权，高毅，张军，吕柳新，夏宁邵．乙肝病毒表面抗原基因在番茄中的表达及免疫活性研究．福建农业大学学报，2002(已录用，排印中)
[136] 马英，林顺权，吕柳新，等．草莓、番木瓜、猕猴桃遗传转化研究初报．园艺学进展(5)，广东科技出版社，2002
[2] Fischhoff DA, Bowdish KS, Perlak FJ, et al. Insect tolerant tomato plants. Biotechnology, 1987, 5:807-813.
[3] 王关林，方宏筠主编．植物基因工程原理与技术．科学出版社，1998
[4] 曹孟良，转基因植物研究及其产业化进展．湖南农业科学，2000(4)：6-8
[5] Daniell H. Environmentally friendly approaches to genetic engineering. In viro Cell. Dev. Biol. Plant Sci. 1999,35,361-168
[6] 范云六，张春义．迎接21世纪农作物生物技术的挑战．生物技术通报，1999(5)：1-6
[7] Dessalegne L, Wetten AC, Caligari PDS. Production of transgenic tomatoes expression oxalateoxidase. Acta Hortic, 1997, 447:457-458.
[8] 付永彩，丁月云，刘新仿，等．抑制衰老的嵌合基因在水稻中的转化．科学通报，1998，43(18)：1963-1967
[9] 黄永芬，汪清胤，付桂荣．美洲拟鲽抗冻蛋白基因(afp)导入番茄的研究．生物化学杂志，1997，13(4)：418-422．
[10] 雷茂良，程金根．全球转基因植物发展现状．生物技术通报，1998(6)：30-32
[11] 贾士荣．转基因植物的环境及食品安全性．生物工程进展，1997，17(6)：36-41
[12] 黄其满．农业生物技术产业发展问题的思考．高技术通讯，2000(6)：107-109
[13] 王锡华，张峰，赵遵田．我国转基因植物研究进展．山东科学，1999(2)：32-36
[14] 王亮，游松．转基因植物的现状．沈阳药科大学学报，2001，18(3)：217-222
[15] Powell Abel P, Nelson RS, De B, et al. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science, 1986, 232:738-743
[16] Whitham S, McCormick S, Baker B.The N gene of tobacco confers resistance to tobacco mosaic virus in transgenic tomato. Proc Natl Acad Sci, 1996,93(16):8776-8781.
[17] 程英豪，吴光，王继伟，等．表达黄瓜花叶病毒外壳蛋白的转基因番茄抗黄瓜花叶病毒浸染．植物学报，1997，39(1)：16-31．
[18] Kunik J, Gafni Y, Citovsky W. Transgenic tomato plants expressing TYLCV capid protein a reresistant to the virus. The role of the nuclear localization signal(NLS) in the resistance. Acta Hortic, 1997, 447:387-391.
[19] Ultzen T, Gielen J, Venenca F, et al. Resistance to tomato spotted with virus in transgenic tomato hybrids. Euphtica, 1995, 85(1-3): 159-168.
[20] 赵淑珍，王昕，王革娇，等．由卫星互补DNA单体和双体基因构建的抗黄瓜花叶病毒的转基因番茄．中国科学(B辑)，1990，(7)：706-713．
[21] Thomzik JZ, Stenzel K, Stocker R. Synthesis of agrapevinephy to a lexinin transgenic tomatos (Lycopersicones-culentum Mill.) conditions resistance against phytophtho rain festans. Physiol Mol Plant Pathol, 1997, 51(4): 265-270.
[22] Jongedij kE, Tiglaar H, Roekel JSC, et al. Synergistic activity of chitimases and β-1,3-glucanases enhances fun-gal resistance in transgenic tomato plants. Euphytica, 1995, 85(1-3): 173-180.
[23] Riely BK, Martin GB. Ancient origin of pathogen recognition specificity conferred by the tomato disease resistance gene Pto. PNAS, 2001, 98: 2059-2064
[24] Vander ST, Bosch D, Hones G, et al. Insect resistance of transgenic plants that express modified Bacillus thuringiensis CryIA(b) and CryIcC genes; a resistance management strategy. Plant Mol Biol, 1994, 26(1):51-59.
[25] Janses S, De Clercq R, Reynaerts A, et al. Green house evaluation of transgenic tomato plants expressing a Bacillus tharingiensis insecticidal crystal protein, for control of Helicoverpaarmigera (Lepidoptera Noctuidae). Meded Fac Land houwwet Univ Gent, 1992, 57(2b): 515-522.
[26] Rhim S-L, Cho HJ. Development of insect resistance in tomato plants expression the δ-endo toxin gene of Bacillus thuringiensis Subsptene brionis. Mol Breed, 1995,1(3): 229-236.
[27] Williamson VM. Root-knot nematode resistance genes in tomato and their potential for future use. Annu. Rev. Phytopathol., 1998,36:277-293
[28] DeBlock M, Botterman J, Vandewiele M, et al. Engineering herbicide rsistance in plants by expression of a detoxifying enzyme. EMBOJ, 1987,6(9):2513-2518.
[29] Benbrook C. Hrbicide resistance: environmental and economic tissues. Proc Bio Expo Butterworth Boston, 1986,(86):27-34.
[30] Fillatti JJ, Haissig B, Mccown B, et al. Development of glyphosate tolerant populus plants through expression of amutant aroagene from salmonellaty phimurium. Basic Life Sci, 1988,44:243-249.
[31] Smith CJS. Inheritance and effect on ripening of antisense polygalacturonase gene in transgenic tomatos. Plant Mol Biol, 1990, 14:369～379
[32] 叶志彪，李汉霞，周国林．番茄多聚半乳糖醛酸反义cDNA克隆的遗传转化与转基因植株再生．园艺学报，1994，21(3)：305-308．
[33] Tucker G, Zhang J. Expression of polygalacturonase and pect inesterase in normal and transgenic tomatoes. Prog Biotechnol, 1996,14:347-354
[34] Oller PW, Min-wong L, Taylor LP, et al. Reversible inhibition of tomato during fruit senescence by antisense RNA. Science, 1991,254:437-439.
[35] 仇润祥，扈延茂，王永胜，等．番茄1-氨基环丙烷羧酸(ACC)合成酶反义RNA-核酶嵌合DNA序列的构建．遗传，1999，1：1-6．
[36] 叶志彪，李汉霞，刘勋甲．利用转基因技术育成耐贮藏番茄——华番1号．中国蔬菜，1999，(1)：6-10．
[37] Klee HJ, Hayford MB, Kretzmer KA, et al. Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants. Plant Cell, 1991,3(11):1187-1193.
[38] Kramer MG, Kellogy J, Wagoner W, et al. Reduced ethylene synthesis and ripening control in tomatoes expressing sad enosy Imethionine hydrolase. NATOASISer, 1997, 34:307-319.
[39] Hightower R, Baden C, Penzes E, et al. Expression of antifreeze proteins in transgenic plants. Plant Mol Biol, 1991,17(5):1013-1021
[40] Penarrubia Loia, Rosalindkim, James Giovannoni. Production of the sweet protein monellin in transgenic plants. Biotechnology, 1994(10): 561-564
[41] Klann M, Hall B, Bennett AB. Antisense acid invertase(T1V1) gene alters soluable sugar composition and size in tomato fruit. Plant Physiol, 1996, 112(3):1321-1330.
[42] Drake CR, Bird CR, Schuch WW. Over expression of phytoenesynthase transgene in tomato plants for enhanced carotenoidcontent. PcT Int Appl WO 9746,690(CI,C12N15/67), 1997-12-11. GBAppl,96/11,981, 1996-06-07, 3.
[43] Bramley PM. The regulation and genetic manipulation of carotenoid
biosynthesis in tomato fruit. Pure Appl Chem, 1997, 69(10) : 2159-2162．
[44] Martineau B, Adams DF, Deverna JW. Ovaryprr dominant expression of acytokin in biosynthetic gene in tomato results in highe totaland soluable fruit solids .Proc Plant Growth Regul Soc Am, 1 994,2 1 : 1 5-24．
[45] Tieman DM, Kausch KD, Serra DM. et al. Field performance oftransgenic tomato with reduced pectin methylesterase activity.Jam Soc Hortic Sci,1995,120(5) :765-768
[46] Mariani C. A chimaeric ribonuclease-inhibitor gene restors fertility to male sterile plants. Nature, 1992,357: 384-387
[47] 张宏，王波．雄性不育嵌合基因的构建及番茄转化研究．遗传，1998,20 (3) : 5-7
[48] Paul, Woeller, LuMin-Wong. Reversible inhibition of tomato of ruit senescence by antisense RNA. Science, 199 1,254:437-439
[49] Bird CR. Using antisense RNA to study gene function inhibition of caroteniod biosynthesis in transgenic tomatoes. Biotechnology, 1991(9) : 635-639
[50] JohnI, DrakeR, FurrellA. Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants molecular and physiological analysis. Plant, 1995, 7(3) :483-490．
[51] 贺竹梅，曹俊，李宝健，等．人促红细胞生成素基因在番茄中的表达．遗传学报,1998, 25 (2) : 155-159
[52] Daniell H. GM crops: public perception and scientific solutions. Trends Pkant Sci. 1999,4,467-469
[53] Matsumoto S, Ikuar K, Ueda M. Characterization of a human glycoprotein (erythropoietin, EPO) produced in cultured tobacco cells, Plant Molecular Biology, 1995,27: 11 63
[54] Mori M, Zhang GH, Kaido M, et al. Efficient production of human gammainterferon in tobacco protoplasts by genetic ally engineer edbrome mosaic virus RNAS. Journal of General Virology,1993,74:1225
[55] Zeitlin L, Olmsted SS, Moench, et al. A humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genitalherpes . Nature Biotechnology, 1998,16:1361
[56] Mason HS, Lam DMK, Arntzen CJ, et al. Expression of hepatitis Bsurface antigen in transgenic plants. Proc. Natl.Acad. Sci. 1992, 89, 1 1745-1 1749
[57] ThanavalaY, Yang YF, Lyons P, et al. Immunogenicity of transgenic plant-derived hepatitisB surface antigen. Proc. Natl. Acad. Sci. 1995,
92,3358-3361
[58] Richter LJ, Thanavala Y, Arntzen CJ, et al. Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat. Biotechnol. 2001,18, 1167-1171
[59] Kapusta J, Modelska A, Figlerowicz M, et al. A plant-derived edible vaccine against hepatitis B virus. FASEB J.1999 ,13, 1796-1799
[60] 刘德虎．利用转基因植物生产药用蛋白．生物技术通报，1999,(4) :1-5
[61] 赵春晖，王荣，赵长生，等．含与不含前导序列的乙肝病毒表面抗原在转基因番茄中表达的研究．农业生物技术学报，2000, 8(2) : 190-193
[62] Haq TA , Mason HS, Clements JD, et al. Oral immunization with arecombinant bacterial antigen produced intransgenic plants. Science, 1995,268,714-716
[63] Lauterslager TGM, Florack DEA, van der Wal TJ, et al.Oral immunisation of naive and primed animals with transgenic potato tubers expressing LT-B. Vaccine, 2001 19,2749-2755
[64] Mason HS, Haq TA, Clements JD 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
[65] Tacket CO, Mason HS, Losonsky G, et al. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat. Med. 1998,4,607-609
[66] Curtiss RI, Cardineau CA. Oral immunisation by transgenetic plants. World Patent Application , WO 1990,90/02484
[67] Tacket CO, Reid RH, Boedeker EC, et al. Enternal immunisation and challenge of volunteers given enterotoxigenic E. coli CFA/II encapsulated in biodegradable microspheres. Vaccine, 1994,12,1270-1274
[68] Streatfield SJ, Jilka JM, Hood EE, et al. Plant-based vaccines: unique advantages. Vaccine, 2001,19, 2742-2748
[69] Arakawa T, Chong DKX, Merritt JL, et al. Expression of choleratoxin B subunit oligomers in transgenic potatoplants. Transgenic Res. 1997, 6, 403-413
[70] Arakawa T, Chong DKX, Langridge WHR, et al. Efficacy of a food plant-basedoral cholera toxin B subunit vaccine. Nat.Biotechnol. 1998,16, 292-297
[71] Mason HS, Ball JM, Shi J-J, Jiang X,et al. Expression of Norwalk virus
capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc. Natl. Acad. Sci. 1996, 93, 5335-5340
[72] Jacket CO, Mason HS, Losonsky G, et al. Human immune responses to a novel Norwalk virus vaccine delivered in transgenic potatoes. J. Infect. Dis. 2000, 182,302-305
[73] McGarvey PB, Hammond J, Dienelt MM, et al. Expression of therabies virus glycoprotein in transgenic tomatoes. Biotechnology, 1995, 13, 1484-1487
[74] YusibvoV, Modelska A, Steplewski K, et al. Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1． Proc. Natl.Acad. Sci. 1997,94,5784-5788
[75] Modelska A, Dietzschold B, Sleysh N, et al. Immunization against rabies with a plant-derived antigen. Proc. Natl.Acad. Sci. 1998,95,2481-2485
[76] SugiyamaY, Hamamoto H, Takemoto S, et al. Systemic production of foreign peptides on the particles surface of tobacco mosaic virus. FEBS Letters, 1995, 359(2-3) ,247-250
[77] Huang Z, Dry I, Webster D, et al. Plant-derived measles virus hemagglutin in protein induces neutralizing antibodies in mice. Vaccine, 2001,19,2163-2171
[78] Tackaberry ES, Dudani AK, Prior F, et al. Development of biopharmaceuticals in plant expression systems: cloning, expression and immunological reactivity of human cytomegalovirus glycoprotein B (UL55) in seeds of transgenic tobacco. Vaccine, 1999, 17, 3020-3029
[79] Turpen TH, Rein! SJ, Charoenvit Y, et al. Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Biotechnology, 1995,13,53-57
[80] Carrillo C, Wigdorovitz A, Oliveros JC, et al. Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants. Journal of General Virology, 1998, .72, 1688-1690
[81] Wigdorovitz A, Carrillo C, Dus Santos MJ, et al. Induction of a protective antibody response to foot and mouth disease in mice following oral or parenteral immunization with alfalfa transgenic plantsexpressing the viral structural protein VP1． Journal of General Virology , 1999 ,255, 347-353
[82] Porta C, Spall VE, Loveland J, et al. Development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides. Journal of General Virology, 1994,202,949-955
[83] Castanon S, Marin MS, Martin-Alonso JM. et al. Immunization with
potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus. Journal of General Virology, 1999,73, 4452-4455
[84] Gomez N, Caxrillo C, Salinas J, et al. Expression of immunogenic glycoprotein S polypeptides from transmissible gastroenteritis coronavirus in transgenic plants. Journal of General Virology, 1998,249, 352-358
[85] Tuboly T, Yu W, Bailey A, et al. Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants. Vaccine, 2000, 18, 2023-2028
[86] Dalsgaard K, Urtenthal A, Jones TD, et al. Plant-derived vaccine protects target animals against a viral disease.Nat. Biotechnol. 1997,15,248-252
[87] Fernandez MR, Martinez-Torrecuadrada JL. Development of an antigen presentation system based on plum pox potyvirus . FEBS Letters, 1998, 27,229-235
[88] 蒋少云，凌均启．转基因番茄防龋疫苗的初步研究．口腔医学纵横杂志，2000, 16 (2) , 94-96
[89] Maygd LS, Panis B, Remy S, et al, Generation of transgenic banana (musa acuminata) plants via agrobacterium-inediated transformation. Biotechnology, 1995, 13, 486-492
[90] Moffat AS. Exploring Transgenic plants as a new vaccine source. Science, 1995, 268(5211 ):658-660
[91] Staub JM, Garcia B, Graves J, et al. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 2000, 18(3) , 333-338
[92] Kim J, Mayfield PS. Protein disulfide isomerase as a regulator of chloroplast translational activation. Science, 1997,278,1954-1957
[93] Reulland E, Miginiac-Maslow M. Regulation of chloroplast enzyme activities by thioredoxins:activation or relief from inhibition. Trends Plant Sci. 1999,4,136-141
[94] De Cosa B, Moar W,Lee SB,et al. Hyper-expression of the Bt Cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat. Biotechnol. 2001, 19(1 ),71-74
[95] Mclnerney TL. Analysis of the ability of five adjuvants to enhance immune responses to a chimeric plant virus displaying an HIV-1 peptide. Vaccine, 1999, 17:11-12,1359-1368
[96] Qingxian Kong, Liz Richter, Yu Fang Yang, et al. Oral immunization with hepatitis B surface antigen expressed in transgenic plants. PNAS, 2001,
98(20) : 11539-11544
[97] Brennan FR. Pseudomonas aeruginosa outer-membrane protein F epitopes are highly immunogenic in mice when expressed on a plant virus. Microbiology, 1999,145:211-220
[98] Henry D, Stephen JS, Keith W. Medical molecular farming:production of antibodies,biopharmaceuticals and edible vaccines in plants. Trends in Plants Science, 2001,6(5) :219-226
[99] Liz JR, Yasmin T, Charles J A. et al. Production of HBsAg in transgenic plants for oral immunization. Nature Biotechnology, 2000,18:1167-1171
[100] Koprowski H, Yusibov V. The green revolution: plants as heterologous expression vectors. Vaccine, 2001,19:2735-2741
[101] Balayan MS, Andjaparidze AG, Savinskaya SS, et al.Evidence for a virus in non-A,non-B hepatitis transmitted via the fecal-oral route. Intervirology, 1983,20(1) :23-31． 3
[102] Reyes GR, Purdy MA, Kim JP, et al. Isolation of cDNA from the virus responsible for enterically transmittednon A, non B hepatitis. Science, 1990,247:1335-1339
[103] Purcell. In; Fields BN, Knipe DM, Howley PM, Chanock RM, Melnick JL, Monooth TP, Roizman B, Strous SE, editors. Fields Virology , 3rd ed, Philadephia: Lippincott-Raven, 1996, 2831-2843
[104] Amanda M Walmsley, Charles J Arntzen. Plants for delivery of edible vaccines. Current Opinion in Biotechnology, 2000,11:126-129
[105] Tsafrir S M, Miguel A G,Kenneth E P. Perspective: edible vaccine-a concept coming of age. Trends in Microbiology, 1998,6(11) :449-453
[106] Mason HS, Tacket CO, Richter LJ, et al. Subunit vaccines produced and delivered in transgenic plants as "edible vaccines". Res. Immunol. 1998,149,71-74
[107] Purdy MA, McCaustland KA, Krawczynski K, et al. Expression of HEV trep E fusion protein containing epitopes recognized by antibodies in sera from human cases and experimentally infected primates. Arch Virol,1992,123:335-349
[108] Wong DC, Purcell RH, Sreenivasan MA, et al.Epidemic and endemic hepatitis in India: evidence for non-A/non-B hepatitis virus etiology. Lancet ,1980,2,876-878
[109] Purdy MA, Karen A, McCaustland, et al. Preliminary evidence that a trp E HEV fusion protein protects cynomolgusmacaques against challenge
with wild type HEV. J Med Virol, 1993, 41:90-94
[110] Li F, Torresi J, Locarnini SA, et al. Amino terminal epitopes are exposed when full length open reading fram 2 of hepatitis E virus is expression in Escherichiacoli, but carbory terminal epitopes are masked. J Med Virol, 1997,52:289-300
[111] Tsarev SA, Tsareva T, Emerson SU, et al. Infeativity titration of prototypestain of hepatitis virus incynomoluss monkeys. J Med Virol, 1994,43:135-147.
[112] Lu FM, Zhuang H, Zhu YH, et al. April iminary study on immune response to hepatitis E virus DNA vaccine in mice. Chinese Med J, 1996,109:919-921
[113] He J, Hoffman SL, Hayes CG, et al. DNA inoculation with a plasmid vector carrying the hepatitis E virus structral protein gene induce immunere sponce in mice. Vaccine, 1997,15(4):357-362
[114] Jameel S, Zafrullah M, Ozdener MH, et al. Expression in animal cells and charactrization of the hepatitis E virus structrul proteins. J Virol, 1996, 70:207-216
[115] 佟玉品，毕胜利，张明程，鲁健，詹美云．戊型肝炎病毒结构区基因ORF2在Pichiapastoris中的分泌表达．中华实验和临床病毒学杂志，2000，14(4)
[116] 灵晓．我国研制成功戊型肝炎疫苗．中华医学杂志，2001，81(2)
[117] 徐道东，赵章忠，王统正等主编．茄果类蔬菜栽培技术，上海科学技术出版社，1999，2
[118] [美]J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯著，金冬雁，黎孟枫等译．分子克隆实验指南，科学出版社，1999，10
[119] 贺竹梅，张绍松．番茄转基因受体系统的研究．应用与环境生物学报，1998，4(3)：247-250
[120] 叶志彪，李汉霞，周国林．番茄子叶离体培养与再生植株．华中农业大学学报，1991，13(3)：291-295
[121] 梁小友，米景九，朱玉贤等．双抗(抗病毒及抗虫)植物表达载体构建及番茄的转化鉴定．植物学报，1994，36(11)：849-854
[122] 王忠华，夏英武．转基因植物中报告基因GUS的表达极其安全性评价．生命科学，2000，12(5)
[123] 马英，张军，林顺权，夏宁邵．利用转基因植物生产基因工程疫苗．厦门大学学报(自然科学版)，2001，40(增刊2)：71—77
[124] 林顺权，宋刚，马英，曾黎辉，吕柳新．果树转基因研究进展．园艺学报，2001，28(增刊)：589-596
[125] Kyle JW, Galvin N, Vogler C, et al. β-glucuronidase assay in animal tissue[A]. In: Gallagher, S Red. Gus Protocols: Using the Gus gene as a reporter gene expression[C]. San Diego: Academic Press, 1992:189-203
[126] Peerbolte R, Leenhouts K, Hooykaas-van Slogteren GMS, et al. Clone from a shooty tobacco crown gall tumor Ⅱ: irregular T-DNA structures and organization T-DNA, methylation and conditional expression of opine genes. Plant Mol Bio, 1986, 7: 285-299.
[127] Finnegan J, McElory D. Transgene inactivation: plants fight back. Biotechnology, 1994, 12:883-888
[128] Stam M, Mof JNM, Kooter JM. The sciences of genes in transgenic plants. Annals of Botany, 1997, 79(1): 3-12
[129] Assaad FF, Tucker KL, Signer ER. Epigenetic repeat-induced gene sciencing (RIGS) in Arabidopsis. Plant Mol Bio, 1993, 22: 1067-1085.
[130] Dorer DR, Henlkoff S. Transgene repeat arrays interact with distant heterochromatin and cause scilencing in cis and trans. Genetics, 1997, 147(3): 1181-1190
[131] Albert H, Dale EC, Lee E, et al. Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J, 1995, 7(4): 649-659
[132] Vergunst AC, Hooykass PJ. Cre/lox-mediated site specific integration of Agrobacterium T-DNA in Arabidopsis thalians by transient expression of cre. Plant Mol Biol, 1998, 38:393-406
[133] Finnegan J, McElroy D. Transgene inactivation: plant fight back, Biotechnology, 1994, 12(9): 883-888
[134] 倪健刚，王立君，应成波，等．转基因番茄快速繁殖若干问题初探．宁波农业科技，1988(3)：2-5
[135] 马英，林顺权，高毅，张军，吕柳新，夏宁邵．乙肝病毒表面抗原基因在番茄中的表达及免疫活性研究．福建农业大学学报，2002(已录用，排印中)
[136] 马英，林顺权，吕柳新，等．草莓、番木瓜、猕猴桃遗传转化研究初报．园艺学进展(5)，广东科技出版社，2002