利用转基因番茄表达人类酸性成纤维细胞生长因子（aFGF）

【作者】 王仁厚；

【导师】 王兴智；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2003， 硕士

【摘要】 迄今为止，在人类中已经发现了22个成纤维细胞生长因子(FGF)超家族成员。成纤维细胞生长因子不但能够促进包括成纤维细胞、内皮细胞、软骨细胞、平滑肌细胞、黑色素细胞等多种细胞的增殖，而且还能够促进脂肪细胞的分化，引发巨噬细胞和成纤维细胞产生白细胞介素6(IL-6)，刺激星形细胞的迁移，延长神经元的寿命。FGF在个体的发育、血管发生、造血、肿瘤发生中发挥重要作用，FGF是通在细胞外与4种FGF酪氨酸受体蛋白激酶相互作用来实现其生物学功能的。人类酸性成纤维细胞生长因子(aFGF，FGF-1)是成纤维细胞生长因子超家族成员。来自于三个胚层的多种细胞都可以表达aFGF。aFGF在治疗帕金森综合症、急性脊柱扭曲性损伤、断指中神经功能重建、脑缺血、肾缺血、心肌梗塞、闭塞性脉管炎、视网膜缺血、胃溃疡及难愈合性伤口等多种临床应用方面具有巨大潜力。 为了满足对aFGF这种价格昂贵的细胞因子的需求，本实验探索利用转基因植物表达aFGF。利用转基因植物大规模生产治疗性蛋白是一种非常有潜力的方法，这是因为此种方法安全、生产成本低、易于操作。我们构建的双元表达载体带有一个经过改构的人类酸性成纤维细胞生长因子基因(afgf)，载体上的选择标记基因为manA，它使得转化过程中可以使用PMI这种不含抗生素及除草剂的选择系统。我们利用农杆菌介导的转化方法获得了afgf转基因番茄植物，并且通过PCR-Southern、RT-PCR证明了afqf在宿主基因组中的整合及表达。更多还原

【Abstract】 To date, in human beings, the fibroblast growth factor (FGF) superfamily consists of 22 members. FGFs can induce proliferation of various types of cells, including fibroblast, endothelial cells, chondrocytes, smooth muscle cells, melanocytes as well as other cells. They can also promote adipocyte differentiation, induce macrophage and fibroblast IL-6 production, stimulate astrocyte migration, and prolong neuronal survival. By acting extracellularly through four tyrosine kinase FGF receptors, FGFs play substantial roles in development, angiogenesis, hematopoiesis, and tumorigenesis. As a member of this superfamily, human acidic fibroblast growth factor (aFGF or FGF1), expressed by a variety of cells from all three germ layers, shows great potential for clinical applications for therapy of a variety of diseases, such as Parkinson’s disease, spinal cord contusion injury, neural regeneration in reimplantation of broken-off finger, brain ischemia, renal ischemia, myocardial infarction, occlusive vascularitis, retinal ischemia, gastric ulcer and nonhealing wound and so on.To meet the increasing demand of this expensive cytokine, we expressed it in transgenic plantSo This is a promising way for producing therapeutic proteins in large scale because of its safety, low expense and ease to manipulate. We constructed a binary vector carrying a modified human afgf gene and a manA gene that allows the use of PMI selection system, an antibiotic and herbicide-free system, during the transformation procedure. We produced transgenic tomato plants by an Agrobacterium tumefacien-mediated method.PCR-Southern blot and RT-PCR revealed integration of afgf gene in the plant genome and successful its transcription in transgenic plant cells.更多还原