口蹄疫病毒基因植物表达载体的构建及其对大豆的遗传转化
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摘要
利用植物生物反应器生产口服疫苗已成为目前研究的热点。与传统疫苗相比，植物源性疫苗具有安全、稳定、高效和廉价等优点。植物作为生产疫苗的载体可将抗原表达于植物的可食部位（例如种子和块茎）。当植物被食用或饲喂时，植物源性疫苗可激发保护性的黏膜免疫反应。随着生物技术的不断完善，植物生物反应器将会成为疫苗生产的有效途径，并部分替代传统疫苗的生产方式。
口蹄疫（Foot and Mouth Disease）是一种高发性流行传染病，常在国际间大规模爆发，目前尚无治愈本病的药物，现在使用较广泛的是酵母源性疫苗。因为酵母源性疫苗生产成本较高，贮运过程中需要低温，注射接种需要特定设备而限制了它的应用。利用转基因植物生产植物源性的口蹄疫疫苗前景广阔。目前，口蹄疫病毒基因（FMDV）已经导入马铃薯、烟草和苜蓿等植物，有关口蹄疫病毒基因导入大豆中的研究未见报道。
根据口蹄疫病毒基因序列FP1（包括FVP1，实验操作与FP1相同）设计引物，同时引入酶切位点BamHI 和SaLI。通过PCR 反应从pGEM-FP1载体上扩增FP1基因，并插入pUCM-T 载体构建成pUCM-FP1中间载体。PCR和酶切鉴定正确的克隆进行测序，序列分析结果表明，插入目标序列完全正确。用BamHI 和SaLI同时酶切pUCM-FP1中间载体和植物组成型表达载体pBin438，构建成植物表达载体pBin-FP1。热激法转化大肠杆菌JM109。经过PCR和酶切鉴定正确的克隆转化根癌农杆菌GV3101，成功构建了2个载体，分别命名为pBin-FP1、pBin-FVP1。用携带此表达载体的根癌农杆菌GV3101侵染大豆。
通过农杆菌介导的大豆子叶节再生体系转化大豆。采用从发芽5-7d的大豆无菌苗切取子叶节外植体，经农杆菌浸染和3d的共培养后, 转移到芽诱导培养基上，10d后从子叶节处诱导出丛生芽。在含50μg/ml卡那霉素的伸长培养基上培养2周后，抗性芽转入生根培养基进行培养，共得到51株卡那抗性植株。28株移栽成活的T0代植株已正常开花和结荚，经PCR检测获得目的基因的阳性植株15株。初步验证了目的基因已整合到大豆基因组中。
Much attention has been focused on the plant-derived oral vaccine production. Compared with the traditional vaccines, the plant-derived oral vaccines are safe, stable, efficient and low-costed. Plant as the delivery vehicle for vaccine can express antigen in edible tissue such as seeds and tubers. The plant-derived vaccine can induce protective mucosal immune response when the transgenic plants are provided as food or feed. With the advances of plant biotechnology, plant bioreactor may become an available approach and will be an alternative means for vaccine production in the future.
Foot and Mouth Disease(FMD) is a highly contagious viral disease, leading to widespread outbreaks in the world. So far there is still none medicine to cure this disease, and the commercial yeast-derived vaccines are widely used. But the high produce cost, cold requirement during store and carry, and requiring specialties when inoculating restrict the utilization of the vaccine. Transgenic plant as plant-derived vaccine is prospect in the production of FMD vaccine. The antigen of FMDV has been transformed into tobacco, potato, and alfalfa et al. but there is no report in soybean yet.
Primers of FMDV-P1(FP1) gene (including FVP1, the procedure same as FP1 gene) were designed according to the nucleotide sequence with the endonuclease recognition site BamHI and SaLI. The FP1 gene was amplified from pGEM-FP1 vector by polymerase chain reaction (PCR) and inserted into the pUCM-T vector constructed to pUCM-FP1. The DNA sequence analysis results showed that the amplified target fragment was correct completely. The pUCM-FP1 vector and plant expression vector pBin438 were digested with corresponding restriction endonuclease and constructed to pBin-FP1. The recombinant plant expression vector was transferred into E.coll JM109. After identification by restriction enzymes and PCR, the correct clone was transferred into Agrobacterium tumefaciens GV3101 by electrotransformation. we constructed two plant expression vectors successfully, named pBin-FP1 and pBin-FVP1. GV3101 which contained the pBin-FP1 and pBin-FVP1 plant expression vectors was used to infect soybean.
The FMDV gene was introduced into soybean with Agrobacterium-cotyledonary node transformation. cotyledonary node explants were prepared from 5-7 day old seedings, infected and co-cultivated with GV3101. Adventitious buds emerged after 10 days on shoot induction medium. the buds were transferred onto elongation medium and selected with 50μg/ml Kanamycine. The shoots were cut off and transferred onto rooting medium when the shoots grew to 3-4 cm high. We abtained 51 kanamycine resistant plants. 27 of them
had already flowered and seeded normally, 15 of which were PCR positive. PCR analysis proved that foreign gene was transferred into soybean genome.
Candidate: Zhu Huiying
Speciality: Botany
Supervisor: Hu Baozhong
Han Tianfu
口蹄疫（Foot and Mouth Disease）是一种高发性流行传染病，常在国际间大规模爆发，目前尚无治愈本病的药物，现在使用较广泛的是酵母源性疫苗。因为酵母源性疫苗生产成本较高，贮运过程中需要低温，注射接种需要特定设备而限制了它的应用。利用转基因植物生产植物源性的口蹄疫疫苗前景广阔。目前，口蹄疫病毒基因（FMDV）已经导入马铃薯、烟草和苜蓿等植物，有关口蹄疫病毒基因导入大豆中的研究未见报道。
根据口蹄疫病毒基因序列FP1（包括FVP1，实验操作与FP1相同）设计引物，同时引入酶切位点BamHI 和SaLI。通过PCR 反应从pGEM-FP1载体上扩增FP1基因，并插入pUCM-T 载体构建成pUCM-FP1中间载体。PCR和酶切鉴定正确的克隆进行测序，序列分析结果表明，插入目标序列完全正确。用BamHI 和SaLI同时酶切pUCM-FP1中间载体和植物组成型表达载体pBin438，构建成植物表达载体pBin-FP1。热激法转化大肠杆菌JM109。经过PCR和酶切鉴定正确的克隆转化根癌农杆菌GV3101，成功构建了2个载体，分别命名为pBin-FP1、pBin-FVP1。用携带此表达载体的根癌农杆菌GV3101侵染大豆。
通过农杆菌介导的大豆子叶节再生体系转化大豆。采用从发芽5-7d的大豆无菌苗切取子叶节外植体，经农杆菌浸染和3d的共培养后, 转移到芽诱导培养基上，10d后从子叶节处诱导出丛生芽。在含50μg/ml卡那霉素的伸长培养基上培养2周后，抗性芽转入生根培养基进行培养，共得到51株卡那抗性植株。28株移栽成活的T0代植株已正常开花和结荚，经PCR检测获得目的基因的阳性植株15株。初步验证了目的基因已整合到大豆基因组中。
Much attention has been focused on the plant-derived oral vaccine production. Compared with the traditional vaccines, the plant-derived oral vaccines are safe, stable, efficient and low-costed. Plant as the delivery vehicle for vaccine can express antigen in edible tissue such as seeds and tubers. The plant-derived vaccine can induce protective mucosal immune response when the transgenic plants are provided as food or feed. With the advances of plant biotechnology, plant bioreactor may become an available approach and will be an alternative means for vaccine production in the future.
Foot and Mouth Disease(FMD) is a highly contagious viral disease, leading to widespread outbreaks in the world. So far there is still none medicine to cure this disease, and the commercial yeast-derived vaccines are widely used. But the high produce cost, cold requirement during store and carry, and requiring specialties when inoculating restrict the utilization of the vaccine. Transgenic plant as plant-derived vaccine is prospect in the production of FMD vaccine. The antigen of FMDV has been transformed into tobacco, potato, and alfalfa et al. but there is no report in soybean yet.
Primers of FMDV-P1(FP1) gene (including FVP1, the procedure same as FP1 gene) were designed according to the nucleotide sequence with the endonuclease recognition site BamHI and SaLI. The FP1 gene was amplified from pGEM-FP1 vector by polymerase chain reaction (PCR) and inserted into the pUCM-T vector constructed to pUCM-FP1. The DNA sequence analysis results showed that the amplified target fragment was correct completely. The pUCM-FP1 vector and plant expression vector pBin438 were digested with corresponding restriction endonuclease and constructed to pBin-FP1. The recombinant plant expression vector was transferred into E.coll JM109. After identification by restriction enzymes and PCR, the correct clone was transferred into Agrobacterium tumefaciens GV3101 by electrotransformation. we constructed two plant expression vectors successfully, named pBin-FP1 and pBin-FVP1. GV3101 which contained the pBin-FP1 and pBin-FVP1 plant expression vectors was used to infect soybean.
The FMDV gene was introduced into soybean with Agrobacterium-cotyledonary node transformation. cotyledonary node explants were prepared from 5-7 day old seedings, infected and co-cultivated with GV3101. Adventitious buds emerged after 10 days on shoot induction medium. the buds were transferred onto elongation medium and selected with 50μg/ml Kanamycine. The shoots were cut off and transferred onto rooting medium when the shoots grew to 3-4 cm high. We abtained 51 kanamycine resistant plants. 27 of them
had already flowered and seeded normally, 15 of which were PCR positive. PCR analysis proved that foreign gene was transferred into soybean genome.
Candidate: Zhu Huiying
Speciality: Botany
Supervisor: Hu Baozhong
Han Tianfu
引文
蔡志强, 徐步进. 转基因植物作为生物反应器生产口蹄疫疫苗的研究进展. 生物工程进展. 2001, 21(4): 8-10
常慧芸, 谢庆阁. 转基因动物生物反应器生产药用蛋白的研究进展.中国兽医科技. 1998, 28(5): 40-42
陈乃用. 实质等同性原则和转基因食品的安全性评价. 工业微生物. 2003, 33 (3): 44-51
程林梅, 孙毅, 岳焕荣. 大豆生物工程研究进展. 大豆科学. 2001, 20(1): 66-70
费蕾, 李晋涛, 吴玉章. 鼠源轮状病毒vp7基因的表达载体构建及转基因植物的研究. 免疫学杂志. 2003, 19 (2): 93-96
韩天富, 王金陵. 大豆开花后光周期反应的研究. 植物学报. 1995, 37 (11): 863-869
韩天富, 王金陵. 中国大豆不同生态类型开花至成熟期对光周期的反应. 作物学报. 1996, 22(1): 20-26
贾建军. 口蹄疫免疫学的一项新发展. 云南畜牧兽医. 1997, 1: 30-31
贾士荣. 转基因食品中标记基因的安全性评价. 中国农业科学. 1997, 30 (2): 1-15
姜鹏斐, 赵启祖, 谢庆阁. 口蹄疫研究进展.中国农业科学. 1998, 32(6): 93-100
李晋涛, 吴玉章, 费蕾, 唐艳, 邹丽云. 鼠源轮状病毒抗原基因的表达载体构建及农杆菌转化研究. 第三军医大学学报. 2002, 24 (8): 892-894
李晓东, 程智慧, 于燕. 转基因植物疫苗的研究现状及前景. 西北农林科技大学学报. 2001, 29(1): 126-128
林福玉, 李宁, 丁翔, 陈永福. 一种制备动物乳腺生物反应器的通用表达载体的构建. 农业生物技术学报. 1998, 6(2): 191-194
刘森, 梁国栋. 利用动物乳腺生物反应器生产药用蛋白. 2000, 16(4): 421-424
刘在新, 赵启祖, 刘卫, 周鹏程, 朱彩珠, 常慧芸, 谢庆阁. 6株口蹄疫病毒结构基因VP1(1D)基因的核苷酸序列分析, 病毒学报. 1998, 14(1): 60-67
柳纪省. 口蹄疫研究进展. 中国兽医科技. 1999, 29(3): 17-22
吕山花, 邱丽娟, 陶波. 转基因植物食品检测技术研究进展. 生物技术通报. 2002, 4 : 34-38
卜云萍, 王广科, 胡国武, 孙红妍, 任勇, 李航, 李明春, 邢来君. 根癌农杆菌介导深黄被孢霉△6-脂肪酸脱氢酶基因转化大豆及其转基因植株再生. 药物生物技术. 2003, 10(5): 273-277
曲志才, 沈大稜. 利用植物生产异源蛋白的研究进展. 生命科学. 2002, 14 (1): 6-8
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 (6): 31-35
沈文涛, 周鹏. 黏膜免疫与转基因植物口服疫苗的研究. 中华预防医学杂志. 2003, 37 (5): 376-378
王凌健, 倪迪安, 陈永宁, 李忠明. 利用转基因胡萝卜表达肺结核疫苗. 植物学报.
2001, 43 (2): 132-137
王新力, 彭学贤. 香蕉果实成熟相关基因ACO1启动子区的克隆及其功能初探. 生物工程学报. 2001, 17 (4): 428-431
魏伟, 钱迎倩, 马克平, 裴克全, 桑卫国. 转基因食品安全性评价的研究进展. 自然资源学报. 2001, 16 (2): 184-190
谢铭. 转基因植物作为生物反应器的新进展. 广西农业科学. 2002, 2: 95-97
徐香玲, 邹联沛, 刘伟华, 李集临. 向大豆导入几丁质酶基因的初步研究. 大豆科学. 1999, 18(2): 101-108
杨广东, 上官小霞, 吴霞, 张林水, 李波, 李燕娥. 选择标记基因剔除及其在转基因棉花中的应用. 棉花学报. 2003, 15 (5): 309-312
杨永钦, 王家驹. 口蹄疫基因工程疫苗研究进展. 云南畜牧兽医. 1999, 1: 27-30
于静, 米凯霞, 秦智伟, 陈晓英. 植物口服疫苗的动物和临床实验. 微生物学通报. 2003, 30 (5) : 132-136
余蓉. 用基因重组技术生产治疗性血浆蛋白制品. 药物生物技术. 1998, 5 (3): 186-192
赵凯, 邹仕平, 张震宇, 郑兆鑫, 徐泉兴, 尤永进, 朱彩珠, 冯霞. 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
周思军, 李希臣, 刘昭军, 刘丽艳, 杨庆凯. 大豆农杆菌介导转化系统的优化研究. 2001, 32(4): 313-319
周思军. 大豆遗传转化的主要障碍及研究进展. 黑龙江农业科学. 2001, 3(60): 24-27
周晓红, 陈晓光, 张晓东, 王亚楠, 李林, 习佳飞, 胡建军. 番茄果实特异性E8 启动子的基因克隆与序列分析. 第一军医大学学报. 2003, 23 (1): 25-28
朱国萍, 徐冲. 植物生物反应器生产绿色疫苗研究进展. 生物工程进展. 2002, 22 (2): 70-73
Arakawa T., Yu J., Chong D.K., Hough J., Engen P.C., and Langridge W.H. A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes. Nature Biotechnol. 1998, 16 (10): 934-938
Carrillo C., Wigdorovitz A., Oliveros J.C., Zamorano P.I., Sadir A.M., Gomez N., Salinas J., Escribano J.M., and Borca M.V. Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants. J.Virol. 1998, 72 (2): 1688-1690
Cheng T.Y., Saka H., and Voqui Dinh T.H. Plant regeneration from soybean cotyledonary node segments in culture. Plant Sci. Lett. 1980, 19: 91-99.
Daniell H., Streatfield S.J., and Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 2001, 6 (5): 219-227
de Wilde C., van Houdt H., de Buck S., Angenon G., de Jaeger G., and Depicker A. Plants as bioreactors for protein production: avoiding the problem of transgene silencing. Plant Mol. Biol. 2000, 43 (2-3): 347-359
Feng Q., Chen X., Ma O., Liu Y., Ding M., Collins R.A., Ko L.S., Xing J., Lau L.T., Yu
A.C., Chen J. Serotype and VP1 gene sequence of a foot-and-mouth disease virus from Hong Kong. Biochem. Biophys. Res. Commun.2003, 302 (4): 715-721
Fischer R., Drossard J., Commandeur U., Schillberg S., and Emans N. Towards molecular farming in the future: moving from diagnostic protein and antibody production in microbes to plants. Biotechnol. Appl. Biochem. 1999, 30 (2): 101-108
Fischer R., Hoffmann K., Schillberg S., and Emans N. Antibody production by molecular farming in plants. J. Biol. Regul. Homeost. Agents. 2000, 14 (2): 83-92
Fujiwara T., Lessard P.A., and Beachy R.N. Seed-specific repression of GUS activity in tobacco plants by antisense RNA. Plant Mol. Biol. 1992, 20 (6): 1059-1069
Geng S., Ma M., Ye H.C., and Li G.F. Anther-specific expression of ipt gene in transgenic tobacco and its effect on plant development. Transgenic Res. 2002, 11 (3): 269-278
Goddijn O.J.M., and Pen J. Plants as bioreactors. Trends Biotechnol. 1995, 13 (9): 379-387
Huang Z., Dry I., Webster D., Strugnell R., and Wesselingh S. Plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice. Vaccine. 2001, 19 (15-16): 2163-2171
Jiang L., and Sun S.S.M. Membrane anchors for vacuolar targeting: application in plant bioreactors, Trends Biotechnol. 2002, 20 (3): 99-102
Kitson J.D., Burke K.L., Pullen L.A., Belsham G.J., Almond J.W. Chimeric polioviruses that include sequences derived from two independent antigenic sites of foot-and-mouth disease virus (FMDV) induce neutralizing antibodies against FMDV in guinea pigs. J .Virol.1991, 65 (6): 3068-3075
Kuvshinov V., Koivu K., Kanerva A., and Pehu E. Molecular control of transgene escape from genetically modified plants. Plant Sci. 2001. 160 (3): 517-522
Larrick J.W., and Thomas D.W. Producing proteins in transgenic plants and animals. Curr. Opin. Biotechnol. 2001, 12 (4): 411-418
Larrick J.W., Yu L., Naftzger C., Jaiswal S., and Wycoff K. Production of secretory IgA antibodies in plants. Biomol. Eng. 2001, 18 (3): 87-94
Liu Y.W., Han C.H., Lee M.H., Hsu F.L., and Hou W.C. Patatin, the tuber storage protein of potato (Solanum tuberosum L.), exhibits antioxidant activity in vitro. J. Agric. Food. Chem. 2003, 51 (15): 4389-4393
Mason H.S., Lam D.M., and Arntzen C.J. Expression of hepatitis B surface antigen in transgenic plants. Proc. Natl. Acad. Sci. USA. 1992, 89 (24): 11745-11749
McGhee J.R., Mestecky J., Dertzbaugh M.T., Eldridge J.H., Hirasawa M., and Kiyono H. The mucosal immune system: from fundamental concepts to vaccine development. Vaccine. 1992, 10 (2): 75-88
McKenna T.S., Rieder E., Lubroth J., Burrage T., Baxt B., Mason P.W. Strategy for producing new foot-and-mouth disease vaccines that display complex epitopes. J.
Biotechnol .1996, 44 (1-3): 83-89
Moffat A.S. Exploring transgenic plants as a new vaccine source. Science. 1995, 268: 658-660
Morcol T.,Akers R.M., Johnson J.L., Williams B.L., Gwazdauskas F.C., Knight J.W., Lubon H., Paleyanda R.K., Drohan W.N., Velander W.H. The porcine mammary gland as a bioreactor for complex proteins. Ann N Y Acad Science. 1994, 721:218-233
Palmiter R.D., Brinster R.L., Hammer R.E., Trumbauer M.E., Rosenfeld M.G., Birnberg N.C., Evans R.M. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growthhormone fusion genes. Nature, 1982, 300 (5893): 611-615
Palmiter R.D., Norstedt G., Gelinas R.E., Hammer R.E., Brinster R.L. Metallothionein-human GH fusion genes stimulate growth of mice. Science . 1983, 222 (4625): 809-814
Peeters K., De Wilde C., De Jaeger G., Angenon G., and Depicker A. Production of antibodies and antibody fragments in plants. Vaccine. 2001, 19 (17-19): 2756-2761
Sala F., Manuela R.M., Barbante A., Basso B., Walmsley A.M., and Castiglione S. Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives. Vaccine. 2003, 21 (7-8): 803-808
Santos M.J.S., Wigdorovitz A., Trono K., Rios R.D., Franzone P.M., Gil F., Moreno J., Carrillo C., Escribano J.M., and Borca M.V. A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine. 2002, 20 (7-8): 1141-1147
Smith M.L., Richter L., Arntzen C.J., Shuler M.L., and Mason H.S. Structural characterization of plant-derived hepatitis B surface antigen employed in oral immunization studies. Vaccine. 2003, 21 (25-26): 4011-4021
Streatfield S.J., Jilka J.M., Hood E.E., Turner D.D., Bailey M.R., Mayor J. M., Woodard S.L., Beifuss K.K., Horn M.E., Delaney D.E., Tizard, I.R. , and howard J.A. Plant–based vaccines: unique advantages. Vaccine. 2001, 19 (17-19): 2742-2748
Thanavala Y., Yang Y.F., Lyons P., Mason H.S., and Arntzen C. Immunogenicity of transgenic plant-derived hepatitis B surface antigen. Proc. Natl. Acad. Sci. USA. 1995, 92 (8): 3358-3361
Tuboly T., Yu W., Bailey A., Degrandis S., Du S., Erickson L., and Nagy E. Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants. Vaccine. 2000, 18 (19): 2023-2028
Twyman R.M., Stoger E., Schillberg S., Christou P., and Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003, 21 (12): 570-578
Usha R., Rohll J.B., Spall V.E., Shanks M., Maule A.J., Johnson J.E., and Lomonossoff G.P. Expression of an animal virus antigenic site on the surface of a plant virus particle.
Virology. 1993, 197 (1): 366-374
Van Cott K.E., Butler S.P., Russell C.G., Subramanian A., Lubon H., Gwazdauskas F.C., Knight J., Drohan W.N., Velander W.H. Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland. Genet Anal. 1999, 15 (3-5): 155-160
Van Cott K.E., Lubon H., Gwazdauskas F.C., Knight J., Drohan W.N., Velander W.H. Recombinant human protein C expression in the milk of transgenic pigs and the effect on endogenous milk immunoglobulin and transferrin levels. Transgenic Res.2001, 10 (1): 43-51
Van Haaren M.J., and Houck C.M. A functional map of the fruit-specific promoter of the tomato 2A11 gene. Plant Mol. Biol. 1993, 21 (4): 625-640
Velander W.H., Page R.L., Morcol T., Russell C.G., Canseco R., Young J.M., Drohan W.N., Gwazdauskas F.C., Wilkins T.D., Johnson J.L. Production of biologically active human protein C in the milk of transgenic mice. Ann N Y Acad Sci. 1992, 665: 391-403
Verch T., Hooper D.C., Kiyatkin A., Steplewski Z., and Koprowski H. Immunization with a plant-produced colorectal cancer antigen. Cancer Immunol. Immunother. 2004, 53 (2): 92-99
Walmsley A.M., and Arntzen C.J. Plants for delivery of edible vaccines. Curr. Opin. Biotechnol. 2000, 11 (2): 126-129
Wright G., Carver A., Cottom D., Reeves D., Scott A., Simons P., Wilmut I., Garner I., Colman A. High level expression of active human alpha-1-antitrypsin in the milk of transgenic sheep. Biotechnology (N Y). 1991, 9: 830-834
Wright G., Cottom D., Cooper J., Dalrymple M., Temperley S., Udell M., Reeves D., Percy J., Scott A. Expression of human alpha 1 antitrypsin in transgenic sheep. Cytotechnology. 1992, 9 (1-3): 77-84
常慧芸, 谢庆阁. 转基因动物生物反应器生产药用蛋白的研究进展.中国兽医科技. 1998, 28(5): 40-42
陈乃用. 实质等同性原则和转基因食品的安全性评价. 工业微生物. 2003, 33 (3): 44-51
程林梅, 孙毅, 岳焕荣. 大豆生物工程研究进展. 大豆科学. 2001, 20(1): 66-70
费蕾, 李晋涛, 吴玉章. 鼠源轮状病毒vp7基因的表达载体构建及转基因植物的研究. 免疫学杂志. 2003, 19 (2): 93-96
韩天富, 王金陵. 大豆开花后光周期反应的研究. 植物学报. 1995, 37 (11): 863-869
韩天富, 王金陵. 中国大豆不同生态类型开花至成熟期对光周期的反应. 作物学报. 1996, 22(1): 20-26
贾建军. 口蹄疫免疫学的一项新发展. 云南畜牧兽医. 1997, 1: 30-31
贾士荣. 转基因食品中标记基因的安全性评价. 中国农业科学. 1997, 30 (2): 1-15
姜鹏斐, 赵启祖, 谢庆阁. 口蹄疫研究进展.中国农业科学. 1998, 32(6): 93-100
李晋涛, 吴玉章, 费蕾, 唐艳, 邹丽云. 鼠源轮状病毒抗原基因的表达载体构建及农杆菌转化研究. 第三军医大学学报. 2002, 24 (8): 892-894
李晓东, 程智慧, 于燕. 转基因植物疫苗的研究现状及前景. 西北农林科技大学学报. 2001, 29(1): 126-128
林福玉, 李宁, 丁翔, 陈永福. 一种制备动物乳腺生物反应器的通用表达载体的构建. 农业生物技术学报. 1998, 6(2): 191-194
刘森, 梁国栋. 利用动物乳腺生物反应器生产药用蛋白. 2000, 16(4): 421-424
刘在新, 赵启祖, 刘卫, 周鹏程, 朱彩珠, 常慧芸, 谢庆阁. 6株口蹄疫病毒结构基因VP1(1D)基因的核苷酸序列分析, 病毒学报. 1998, 14(1): 60-67
柳纪省. 口蹄疫研究进展. 中国兽医科技. 1999, 29(3): 17-22
吕山花, 邱丽娟, 陶波. 转基因植物食品检测技术研究进展. 生物技术通报. 2002, 4 : 34-38
卜云萍, 王广科, 胡国武, 孙红妍, 任勇, 李航, 李明春, 邢来君. 根癌农杆菌介导深黄被孢霉△6-脂肪酸脱氢酶基因转化大豆及其转基因植株再生. 药物生物技术. 2003, 10(5): 273-277
曲志才, 沈大稜. 利用植物生产异源蛋白的研究进展. 生命科学. 2002, 14 (1): 6-8
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 (6): 31-35
沈文涛, 周鹏. 黏膜免疫与转基因植物口服疫苗的研究. 中华预防医学杂志. 2003, 37 (5): 376-378
王凌健, 倪迪安, 陈永宁, 李忠明. 利用转基因胡萝卜表达肺结核疫苗. 植物学报.
2001, 43 (2): 132-137
王新力, 彭学贤. 香蕉果实成熟相关基因ACO1启动子区的克隆及其功能初探. 生物工程学报. 2001, 17 (4): 428-431
魏伟, 钱迎倩, 马克平, 裴克全, 桑卫国. 转基因食品安全性评价的研究进展. 自然资源学报. 2001, 16 (2): 184-190
谢铭. 转基因植物作为生物反应器的新进展. 广西农业科学. 2002, 2: 95-97
徐香玲, 邹联沛, 刘伟华, 李集临. 向大豆导入几丁质酶基因的初步研究. 大豆科学. 1999, 18(2): 101-108
杨广东, 上官小霞, 吴霞, 张林水, 李波, 李燕娥. 选择标记基因剔除及其在转基因棉花中的应用. 棉花学报. 2003, 15 (5): 309-312
杨永钦, 王家驹. 口蹄疫基因工程疫苗研究进展. 云南畜牧兽医. 1999, 1: 27-30
于静, 米凯霞, 秦智伟, 陈晓英. 植物口服疫苗的动物和临床实验. 微生物学通报. 2003, 30 (5) : 132-136
余蓉. 用基因重组技术生产治疗性血浆蛋白制品. 药物生物技术. 1998, 5 (3): 186-192
赵凯, 邹仕平, 张震宇, 郑兆鑫, 徐泉兴, 尤永进, 朱彩珠, 冯霞. 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
周思军, 李希臣, 刘昭军, 刘丽艳, 杨庆凯. 大豆农杆菌介导转化系统的优化研究. 2001, 32(4): 313-319
周思军. 大豆遗传转化的主要障碍及研究进展. 黑龙江农业科学. 2001, 3(60): 24-27
周晓红, 陈晓光, 张晓东, 王亚楠, 李林, 习佳飞, 胡建军. 番茄果实特异性E8 启动子的基因克隆与序列分析. 第一军医大学学报. 2003, 23 (1): 25-28
朱国萍, 徐冲. 植物生物反应器生产绿色疫苗研究进展. 生物工程进展. 2002, 22 (2): 70-73
Arakawa T., Yu J., Chong D.K., Hough J., Engen P.C., and Langridge W.H. A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes. Nature Biotechnol. 1998, 16 (10): 934-938
Carrillo C., Wigdorovitz A., Oliveros J.C., Zamorano P.I., Sadir A.M., Gomez N., Salinas J., Escribano J.M., and Borca M.V. Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants. J.Virol. 1998, 72 (2): 1688-1690
Cheng T.Y., Saka H., and Voqui Dinh T.H. Plant regeneration from soybean cotyledonary node segments in culture. Plant Sci. Lett. 1980, 19: 91-99.
Daniell H., Streatfield S.J., and Wycoff K. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 2001, 6 (5): 219-227
de Wilde C., van Houdt H., de Buck S., Angenon G., de Jaeger G., and Depicker A. Plants as bioreactors for protein production: avoiding the problem of transgene silencing. Plant Mol. Biol. 2000, 43 (2-3): 347-359
Feng Q., Chen X., Ma O., Liu Y., Ding M., Collins R.A., Ko L.S., Xing J., Lau L.T., Yu
A.C., Chen J. Serotype and VP1 gene sequence of a foot-and-mouth disease virus from Hong Kong. Biochem. Biophys. Res. Commun.2003, 302 (4): 715-721
Fischer R., Drossard J., Commandeur U., Schillberg S., and Emans N. Towards molecular farming in the future: moving from diagnostic protein and antibody production in microbes to plants. Biotechnol. Appl. Biochem. 1999, 30 (2): 101-108
Fischer R., Hoffmann K., Schillberg S., and Emans N. Antibody production by molecular farming in plants. J. Biol. Regul. Homeost. Agents. 2000, 14 (2): 83-92
Fujiwara T., Lessard P.A., and Beachy R.N. Seed-specific repression of GUS activity in tobacco plants by antisense RNA. Plant Mol. Biol. 1992, 20 (6): 1059-1069
Geng S., Ma M., Ye H.C., and Li G.F. Anther-specific expression of ipt gene in transgenic tobacco and its effect on plant development. Transgenic Res. 2002, 11 (3): 269-278
Goddijn O.J.M., and Pen J. Plants as bioreactors. Trends Biotechnol. 1995, 13 (9): 379-387
Huang Z., Dry I., Webster D., Strugnell R., and Wesselingh S. Plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice. Vaccine. 2001, 19 (15-16): 2163-2171
Jiang L., and Sun S.S.M. Membrane anchors for vacuolar targeting: application in plant bioreactors, Trends Biotechnol. 2002, 20 (3): 99-102
Kitson J.D., Burke K.L., Pullen L.A., Belsham G.J., Almond J.W. Chimeric polioviruses that include sequences derived from two independent antigenic sites of foot-and-mouth disease virus (FMDV) induce neutralizing antibodies against FMDV in guinea pigs. J .Virol.1991, 65 (6): 3068-3075
Kuvshinov V., Koivu K., Kanerva A., and Pehu E. Molecular control of transgene escape from genetically modified plants. Plant Sci. 2001. 160 (3): 517-522
Larrick J.W., and Thomas D.W. Producing proteins in transgenic plants and animals. Curr. Opin. Biotechnol. 2001, 12 (4): 411-418
Larrick J.W., Yu L., Naftzger C., Jaiswal S., and Wycoff K. Production of secretory IgA antibodies in plants. Biomol. Eng. 2001, 18 (3): 87-94
Liu Y.W., Han C.H., Lee M.H., Hsu F.L., and Hou W.C. Patatin, the tuber storage protein of potato (Solanum tuberosum L.), exhibits antioxidant activity in vitro. J. Agric. Food. Chem. 2003, 51 (15): 4389-4393
Mason H.S., Lam D.M., and Arntzen C.J. Expression of hepatitis B surface antigen in transgenic plants. Proc. Natl. Acad. Sci. USA. 1992, 89 (24): 11745-11749
McGhee J.R., Mestecky J., Dertzbaugh M.T., Eldridge J.H., Hirasawa M., and Kiyono H. The mucosal immune system: from fundamental concepts to vaccine development. Vaccine. 1992, 10 (2): 75-88
McKenna T.S., Rieder E., Lubroth J., Burrage T., Baxt B., Mason P.W. Strategy for producing new foot-and-mouth disease vaccines that display complex epitopes. J.
Biotechnol .1996, 44 (1-3): 83-89
Moffat A.S. Exploring transgenic plants as a new vaccine source. Science. 1995, 268: 658-660
Morcol T.,Akers R.M., Johnson J.L., Williams B.L., Gwazdauskas F.C., Knight J.W., Lubon H., Paleyanda R.K., Drohan W.N., Velander W.H. The porcine mammary gland as a bioreactor for complex proteins. Ann N Y Acad Science. 1994, 721:218-233
Palmiter R.D., Brinster R.L., Hammer R.E., Trumbauer M.E., Rosenfeld M.G., Birnberg N.C., Evans R.M. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growthhormone fusion genes. Nature, 1982, 300 (5893): 611-615
Palmiter R.D., Norstedt G., Gelinas R.E., Hammer R.E., Brinster R.L. Metallothionein-human GH fusion genes stimulate growth of mice. Science . 1983, 222 (4625): 809-814
Peeters K., De Wilde C., De Jaeger G., Angenon G., and Depicker A. Production of antibodies and antibody fragments in plants. Vaccine. 2001, 19 (17-19): 2756-2761
Sala F., Manuela R.M., Barbante A., Basso B., Walmsley A.M., and Castiglione S. Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives. Vaccine. 2003, 21 (7-8): 803-808
Santos M.J.S., Wigdorovitz A., Trono K., Rios R.D., Franzone P.M., Gil F., Moreno J., Carrillo C., Escribano J.M., and Borca M.V. A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine. 2002, 20 (7-8): 1141-1147
Smith M.L., Richter L., Arntzen C.J., Shuler M.L., and Mason H.S. Structural characterization of plant-derived hepatitis B surface antigen employed in oral immunization studies. Vaccine. 2003, 21 (25-26): 4011-4021
Streatfield S.J., Jilka J.M., Hood E.E., Turner D.D., Bailey M.R., Mayor J. M., Woodard S.L., Beifuss K.K., Horn M.E., Delaney D.E., Tizard, I.R. , and howard J.A. Plant–based vaccines: unique advantages. Vaccine. 2001, 19 (17-19): 2742-2748
Thanavala Y., Yang Y.F., Lyons P., Mason H.S., and Arntzen C. Immunogenicity of transgenic plant-derived hepatitis B surface antigen. Proc. Natl. Acad. Sci. USA. 1995, 92 (8): 3358-3361
Tuboly T., Yu W., Bailey A., Degrandis S., Du S., Erickson L., and Nagy E. Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants. Vaccine. 2000, 18 (19): 2023-2028
Twyman R.M., Stoger E., Schillberg S., Christou P., and Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003, 21 (12): 570-578
Usha R., Rohll J.B., Spall V.E., Shanks M., Maule A.J., Johnson J.E., and Lomonossoff G.P. Expression of an animal virus antigenic site on the surface of a plant virus particle.
Virology. 1993, 197 (1): 366-374
Van Cott K.E., Butler S.P., Russell C.G., Subramanian A., Lubon H., Gwazdauskas F.C., Knight J., Drohan W.N., Velander W.H. Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland. Genet Anal. 1999, 15 (3-5): 155-160
Van Cott K.E., Lubon H., Gwazdauskas F.C., Knight J., Drohan W.N., Velander W.H. Recombinant human protein C expression in the milk of transgenic pigs and the effect on endogenous milk immunoglobulin and transferrin levels. Transgenic Res.2001, 10 (1): 43-51
Van Haaren M.J., and Houck C.M. A functional map of the fruit-specific promoter of the tomato 2A11 gene. Plant Mol. Biol. 1993, 21 (4): 625-640
Velander W.H., Page R.L., Morcol T., Russell C.G., Canseco R., Young J.M., Drohan W.N., Gwazdauskas F.C., Wilkins T.D., Johnson J.L. Production of biologically active human protein C in the milk of transgenic mice. Ann N Y Acad Sci. 1992, 665: 391-403
Verch T., Hooper D.C., Kiyatkin A., Steplewski Z., and Koprowski H. Immunization with a plant-produced colorectal cancer antigen. Cancer Immunol. Immunother. 2004, 53 (2): 92-99
Walmsley A.M., and Arntzen C.J. Plants for delivery of edible vaccines. Curr. Opin. Biotechnol. 2000, 11 (2): 126-129
Wright G., Carver A., Cottom D., Reeves D., Scott A., Simons P., Wilmut I., Garner I., Colman A. High level expression of active human alpha-1-antitrypsin in the milk of transgenic sheep. Biotechnology (N Y). 1991, 9: 830-834
Wright G., Cottom D., Cooper J., Dalrymple M., Temperley S., Udell M., Reeves D., Percy J., Scott A. Expression of human alpha 1 antitrypsin in transgenic sheep. Cytotechnology. 1992, 9 (1-3): 77-84