公开(公告)号：WO1998021348A1

公开(公告)日：1998-05-22

申请号：PCT/US1997020603

申请日：1997-11-12

Applicant: BATTELLE MEMORIAL INSTITUTE

Inventor： BATTELLE MEMORIAL INSTITUTE ,  HOOKER, Brian, S. ,  DAI, Ziyu ,  GAO, Jianwei ,  KINGSLEY, Mark, T. ,  WELLER, Richard, E.

IPC: C12N15/82

CPC classification number: C12N15/8257 ,  A61K38/00 ,  C07K14/485 ,  C07K2319/00

Abstract: The production of hEGF is achieved in both whole plants and plant cell culture wherein the hEGF has a length of at least 200 amino acids. For epidermal growth factor this would comprise at least a tetramer of EGF units. Effectiveness or production of the translation process has been increased according to the present invention by (1) cloning of pre-pro-EGF cDNA of approximately 4.5 kb into both whole plants and cell culture to increase overall titers of active hEGF; (2) synthesizing cDNA and transforming plants and cell culture for production of an oligomeric polypeptide consisting of repeated hEGF domains; and (3) increasing the overall size of the gene to be expressed with a fusion construct encoding hEGF linked to a protein that is efficiently produced in plant systems. As needed, synthetic cDNA includes plant-specific proteolytic cleavage sites between EGF repeats to facilitate correct processing in planta. Appropriate proteolytic cleavage sites upstream and downstream of hEGF are added if needed to obtain final product. In whole plants, use of a regulatory element confers hEGF production characteristics into traditionally non-saleable portions of crop plants, such as the leafy tops of potatoes. Use of potato tops under post-harvest conditions, results in overexpression production of hEGF in non-saleable plant portions towards the end of the harvesting season, without affecting crop quality.

Abstract translation: 在整个植物和植物细胞培养物中实现hEGF的产生，其中hEGF具有至少200个氨基酸的长度。 对于表皮生长因子，这将包括至少四聚体的EGF单元。 根据本发明，翻译过程的有效性或生产已经通过（1）将大约4.5kb的前原EGF cDNA克隆到整个植物和细胞培养物中以增加活性hEGF的总滴度; （2）合成cDNA和转化植物和细胞培养物以产生由重复的hEGF结构域组成的寡聚多肽; 和（3）增加用编码与在植物系统中有效产生的蛋白质连接的hEGF的融合构建体来表达基因的总体大小。 根据需要，合成cDNA包括EGF重复之间的植物特异性蛋白水解切割位点，以促进植物中的正确加工。 如果需要获得最终产品，则加入hEGF上游和下游的合适的蛋白水解切割位点。 在整个植物中，使用调节元件将hEGF生产特征赋予作物植物的传统不可销售部分，例如马铃薯的叶状顶部。 在收获后的条件下使用马铃薯盖，导致在收获季节结束时在非销售植物部分过度表达hEGF，而不影响作物品质。