Development of a novel procedure for production of an antibody protein against human Hepatitis virus in transgenic plants

开发在转基因植物中生产抗人肝炎病毒抗体蛋白的新方法

• 批准号: 12556003
• 负责人: SHIMADA Hiroaki
• 金额: $ 3.9万
• 依托单位: Tokyo University of Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12556003/
• 关键词: sugarcane; biomass; molecular farming; transgenic plant; sucrose synthase; model system; Hepatitis virus; antibody; B型肝炎ウィルス抗体; 植物工場; 水モミ; SPK; 組換え体イネ; HBs結合活性; 篩部特異的タンパク質; 遺伝子発現; レポーター; 形質転換

Molecular Farming is the genetic engineering of plants to produce totally new-use, non-food products, such as biopharmaceuticals. We selected sugarcane as a plant material because sugarcane is hung plant and may be a suitable plant for future molecular farming. Sugarcane callus was established from meristem tissues and propagated. Agrobacterium and particle gun methods were applied for a transformation. In sink organs, sucrose is metabolized by sucrose synthase as an initial step in biosynthesis of storage products. Rice SPK, a CDPK, was unique to the endosperm of immature seeds. It was suggested that activity of sucrose synthese is modulated by SPK for the biosynthesis of storage substances in immature seeds. Rpp16 and Rpp17 genes specifically expressed in phloem tissue were isolated. To isolate the genes expressed in the phboem tissues, wedeveloped a new method for limitation isolation of the specific cells using laser capture microdissection. By this method, we isolated several useful genes. We tried to establish a model system for producing protein in a transgenic plant. As a model compound, a broad-spectrum antimicrobial peptide, thanatin, was used. A crude extract from leaves of the rice transformant showed apparent antimicrobial activity as an active form. It aimed at manifesting human antibody (KF94) with the neutralization activity for hepatitis A virus (HVA) in the green leaf of rice plant. We constructed an artificial gene with/without signal sequence of immuno globulin or KDEL signal of ER. They were introduced into rice plant and detected expression of each antibody gene: L chain or H chain. It seems that they would be applicable to create a rice plant manifesting the functional human Fab fragment by crossing these rice plant which have independently manifested L chain and H chain

分子农业是植物的基因工程，以生产全新用途的非食品产品，如生物制药。我们选择甘蔗作为植物材料，因为甘蔗是悬挂植物，可能是未来分子农业的合适植物。从甘蔗茎尖组织中建立愈伤组织并进行增殖。农杆菌法和粒子枪法用于转化。在库器官中，蔗糖通过蔗糖合成酶代谢，作为贮藏产物生物合成的起始步骤。水稻SPK是水稻未成熟种子胚乳所特有的一种CDPK。说明SPK对蔗糖合成活性的调节与未成熟种子贮藏物质的合成有关。Rpp16和Rpp17基因在韧皮部组织中特异表达。为了分离组织中表达的基因，我们发展了一种新的激光捕获显微切割有限分离特定细胞的方法。通过这种方法，我们分离了几个有用的基因。我们试图建立一个在转基因植物中生产蛋白质的模式系统。作为模型化合物，使用广谱抗菌肽thanatin。从水稻叶中提取的粗提物作为活性形式显示出明显的抗菌活性。目的是在水稻植株的绿色叶片中显示具有中和甲型肝炎病毒（HVA）活性的人源抗体（KF 94）。我们构建了一个带有或不带有免疫球蛋白信号序列或ER的KDEL信号的人工基因。将它们导入水稻植株中，检测每个抗体基因：L链或H链的表达。通过杂交这些具有独立表现的L链和H链的水稻植物，它们似乎适用于产生表现功能性人Fab片段的水稻植物

项目成果

H.Kuroda, N.Takahashi, H.Shimada, M.Seki, K.Shinozaki, M.Matsui: "Classification and expression of Arabidopsis F-bo-containing protein genes"Plant Cell Physiology. 43. 1073-1085 (2002)

H.Kuroda、N.Takahashi、H.Shimada、M.Seki、K.Shinozaki、M.Matsui：“含拟南芥 F-bo 的蛋白质基因的分类和表达”植物细胞生理学。

T.Tuge,N.Inagaki,T.Yoshizumi,H.Shimada,T.Kawamoto,R.Matsuki,M.Matsui: "Phytochrome-mediated control of COP1 gene expression in rice plants"Mol.Gen.Genet.. (印刷中).

T.Tuge、N.Inagaki、T.Yoshizumi、H.Shimada、T.Kawamoto、R.Matsuki、M.Matsui：“光敏色素介导的水稻 COP1 基因表达控制”Mol.Gen.Genet..（打印期间）。

T.Asano, N.Kunieda, Y.Omura, T.Kawasaki, M.Takano, M.Sato, H.Furuhashi, T.Mujin, F.Takaiwa, C.Wu, T.Tada, T.Satozawa, M.Sakamoto, H.Shimada: "Rice SPK, a calmodulin-like domain protein kinase, is required for storage product accumulation during seed devel

T.Asano、N.Kunieda、Y.Omura、T.Kawasaki、M.Takano、M.Sato、H.Furuhashi、T.Mujin、F.Takaiwa、C.Wu、T.Tada、T.Satozawa、M.

Takayuki Asano, Noriko Kunieda, Yuhi Omura, Tsutomu Kawasaki, Makoto Takaiwa, Chuan-yin Wu, Yuichi Tada, Tomomi Satozawa, Masahiro Sakamoto, Hiroaki Shimada: "Rice SPK, a calmodulin-like domain protein kinase, is required for storage product accumulation

Takayuki Asano、Noriko Kunieda、Yuhi Omura、Tsutomu Kawasaki、Makoto Takaiwa、Chuan-yin Wu、Yuichi Tada、Tomomi Satozawa、Masahiro Sakamoto、Hiroaki Shimada：“Rice SPK 是一种钙调蛋白样结构域蛋白激酶，是储存产物积累所必需的

浅野敬幸,島田浩章,高野誠,佐藤美帆,久保中央,西口正通,門脇光一: "イネにおけるカボチャ師部タンパク質遺伝子ホモログの発現解析"育種学研究. 2(別1). 68-68 (2000)

Takayuki Asano，Hiroaki Shimada，Makoto Takano，Miho Sato，Chuo Kubo，Masamichi Nishiguchi，Koichi Kadowaki：“水稻韧皮部蛋白基因同源物的表达分析”育种研究2（第1部分）。