导　　师： 赵德刚

学科专业： G1010

授予学位： 硕士

作　　者： ;

机构地区： 贵州大学

摘　　要： 人表皮生长因子(hegf)，是一种很强的促细胞有丝分裂原，微量的hegf能强烈地促进细胞的分裂和生长，在临床上可用于治疗烧伤、创伤、角膜移植等，在保护消化道上皮细胞及修复粘膜细胞中发挥巨大作用，hegf还可用于化妆品，由于其特殊的生物学效应使其具有很大的潜在经济价值。转基因植物作为表达药用蛋白的反应器，与传统的生产方法相比，具有许多优点，如较低的生产成本、快速规模化的生产模式、不存在对人体健康有害的病原体，同时具有折叠和正确组装复合蛋白的能力等，已成为目前植物基因工程研究热点之一。 本论文研究根据己发表的人表皮生长因子序列和植物密码子偏爱性，设计并人工合成了hegf基因序列，为提高其表达效率，在该基因的3’端设计了gpgp连接杆和内质网定位序列sekdel。构建了人表皮生长因子基因的组成型植物表达载体psh-hegfs，包含报告基因gus、目的基因hegf—sekdel、抗性选择标记基因为nptii，启动子为35s，终止子为nos。构建了冷诱导启动子启动删除系统的植物表达载体pfcor15fgn；冷诱导启动子启动删除系统、且含有hegf基因的植物表达载体pfcor15fgn—hegfs；以及适合单子叶植物遗传转化的、含有hegf基因的植物表达载体pcambia-pnos-pnn—hegfs等。并采用农杆菌介导法，将植物表达载体psh-hegfs遗传转化烟草和矮牵牛等，通过抗性筛选、gus报告基因的组织化学检测以及hegf基因表达元件的pcr分子检测，证明hegf基因已经成功整合到烟草和矮牵牛的基因组中，成功获得了含有hegf基因的转基因烟草和矮牵牛植株，为进一步研究植物表达人表皮生长因子的生物学功能，研制以转基因植物为生物反应器生产人表皮生长因子等药物奠定了基础。 The human epidermal growth factor /(hEGF/) is a powerful mitogen for a wide variety of cells in culture and in vivo, which is a kind of tyrosine kinase leading to itself phosphorylation and cells proliferation. It is often applied to treat burns, scald, cornea transplant, and intensively promotes the proliferation and growth at very low dosage in many types of cells especially in protecting and repairing the alimentary canal epithelium cells and wound healing. Furthermore, the human EGF can be also widely used in the cosmetic. It has big latency economic value because it has special and potential biological effects. Plants have many advantages compared with traditional systems for the production of biopharmaceutical proteins. These include the low cost of production, rapid scalability, the absence of human pathogens and the ability to fold and assemble complex proteins accurately.It becomes one of the research hotspots. In the present study, The hEGF gene sequence has been designed and chemically synthesized according to enregistered hEGF sequence and the codons bias in plant. Amino acid GPGP and SEKDEL as endoplasmic reticulum orientation sequence were added in 3' end, in order to improve protein expression. Expression vectors pSH-hEGFS、 pFCor15FGN、 pCambia-Pnos-PNN-hEGFS and pFCor15FGN-hEGF suitable for expression in plant has been constructed. The CaMV35S promoter and NOS terminator fragments were syncretized with reporter GUS gene and targeted gene. The selectable maker gene was neomycin phosphotrasferase gene, which allowed selection with kanamycin. Both tobacco and petunia were been transformed by Agrobacterium tumefaciens . The buds were selected by kanamycin. The results of GUS histochemical tinction and hEGF gene PCR indicated that hEGF gene have been integrased into genomes of tobacco and petunia respectively help to develop the bioactor producing hEGF using transgenic plants.

关 键 词： 表皮生长因子 植物表达载体 转基因植物 遗传转化 农杆菌介导 植物基因工程

分 类 号： [Q943.2]

领　　域： [生物学]