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Production of monoclonal antibodies in eggs of transgenic avians

转基因禽蛋中单克隆抗体的生产

基本信息

批准号：
14350434
负责人：
KAMIHIRA Masamichi
金额：
$ 10.62万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14350434/
关键词：
transgenic avian monoclonal antibody retroviral vector scFv-Fc animal cell chimeric antibody chicken quail

项目摘要

Transgenic chickens have been proposed as a transgenic bioreactor for the production of recombinant proteins. To date, although various procedures for generating transgenic avians have been reported, the expression of a transgene at a commercially feasible level has not been attained. We used a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus(MoMLV) pseudotyped with vesicular stomatitis virus G protein(VSV-G) for gene introduction to avian embryos. For the generation of transgenic chickens producing monoclonal antibodies, we firstly constructed a retroviral vector plasmid for the expression of a single-chain antibody fragment(scFv), derived from an anti-prion protein monoclonal antibody, fused with the Fc region of human IgG1, as a model antibody. We steadily generated transgenic chickens (GO) by injecting a concentrated retroviral vector into the heart of developing embryos after 55 h incubation and followed by embryo culture to hatch. The GO ch … More ickens expressed the transgene product in their serum and eggs at high levels (~5.6 mg/ml) throughout the breeding period. The expression level in egg white corresponds to almost one tenth that of ovalbumin, a major protein in egg white, and more than equivalent to that of lysozyme. Furthermore, we could obtain G1 and G2 transgenic progeny derived from a GO chicken. The transgenic progeny also expressed the transgene product in their serum and eggs. Next, we constructed a retroviral vector for the production of a chimeric anti-CD2 antibody, in which the bicistronic expression of H-and L-chain genes of the antibody was mediated by an internal ribosomal entry site(IRES) sequence derived from encephalomyocarditis virus(EMCV). The viral vector was injected to chicken embryos after 55 h incubation to generate GO transgenic chickens. The chickens hatched expressed anti-CD2 antibody in the serum and eggs. This system demonstrates the reality of a transgenic chicken bioreactor for the commercial production of monoclonal antibodies. Less

转基因鸡已被提议作为生产重组蛋白的转基因生物反应器。迄今为止，尽管已经报道了用于产生转基因禽类的各种方法，但是还没有达到商业上可行的水平的转基因表达。我们使用了复制缺陷型泛嗜性逆转录病毒载体的基础上莫洛尼鼠白血病病毒（MoMLV）假型水泡性口炎病毒G蛋白（VSV-G）的基因导入禽胚胎。为了获得产生单克隆抗体的转基因鸡，我们首先构建了用于表达单链抗体片段（scFv）的逆转录病毒载体质粒，所述单链抗体片段（scFv）来源于抗朊蛋白单克隆抗体，与人IgG 1的Fc区融合，作为模型抗体。我们通过将浓缩的逆转录病毒载体注射到孵育55小时后发育中的胚胎的心脏中，然后通过胚胎培养孵化来稳定地产生转基因鸡（GO）。GO CH ...更多信息在整个繁殖期间，ickens在其血清和卵中以高水平（~5.6mg/ml）表达转基因产物。在卵白色中的表达水平几乎相当于卵清蛋白（卵白色中的主要蛋白质）的十分之一，并且超过溶菌酶的表达水平。此外，我们可以获得G1和G2代转基因后代的GO鸡。转基因后代也在其血清和卵中表达转基因产物。接下来，我们构建了一个逆转录病毒载体的嵌合抗CD2抗体的生产，其中的H-和L-链基因的抗体的双顺反子表达介导的内部核糖体进入位点（IRES）序列来自脑心肌炎病毒（EMCV）。将病毒载体注射到孵育55 h后的鸡胚中以产生GO转基因鸡。孵化出的雏鸡血清和蛋中均表达抗CD2抗体。该系统展示了用于商业化生产单克隆抗体的转基因鸡生物反应器的现实。少

项目成果

期刊论文数量（18）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

上平正道, 西島謙一, 飯島信司: "金の卵を産むニワトリをつくる-トランスジェニック鳥類を使った有用タンパク質の生産"化学と生物. 41(2). 70-72 (2003)

Masamichi Kamihira、Kenichi Nishijima、Shinji Iijima：“创造能下金蛋的鸡——利用转基因鸟类生产有用的蛋白质”，化学与生物学 41(2) (2003)。

DOI：
发表时间：
期刊：
影响因子：
0
作者：
通讯作者：

Production of pharmaceutical proteins by transgenic avian

转基因禽类生产药用蛋白

DOI：
发表时间：
2004
期刊：
Animal Cell Technology : Basic & Applied Aspects(Kluwer Academic Publishers) Vol.13
影响因子：
0
作者：
A.Hotta;K.Ono;S.Mizuarai;K.Yamaguchi;K.Nishijima;M.Kamihira;S.Iijima
通讯作者：
S.Iijima

Animal Cell Technology : Basic & Applied Aspects, Vol.13(Production of pharmaceutical proteins by transgenic avian)

动物细胞技术：基础