人表皮生长因子在烟草中的表达及其重组蛋白的初步鉴定

Expression of human epidermal growth factor in tobacco plants and primary identification of the recombinant protein

目的:运用Dot-ELISA方法对转基因烟草中所表达的重组hEGF蛋白进行初步检测,以确定是否表达出具有免疫功能的蛋白质。方法:将所合成的hEGF基因置于CAMV35S启动子的驱动下,用农杆菌介导的方法转入烟草中,经过抗性筛选和PCR鉴定出阳性转化子后,提取转化子的总蛋白。总蛋白提取物经透析后,取适当浓度蛋白质点在尼龙膜上,用Dot-ELISA方法对所提取的蛋白质进行测定。结果:结果表明,转基因烟草中的重组蛋白能与抗体发生特异性结合,Dot-ELISA反应后的膜上显示出清晰的棕色斑点。结论:通过实验证明Dot-ELISA方法是一种简便快捷的检测方法,适用于植物基因工程中大规模重组蛋白生产的初步检测。

Objective： To identify the immunity activity of recombinant hEGF with Dot ELISA method in hEGF gene transgenic tobacco plants, Methods： In this study, the synthetic hEGF gene was controlled under CAMV35S promoter and was transformed into tobacco plants via agrobacterium-mediated procedure. Total protein was extracted from the transforms identified by PCR, and extraction was used to test the immunity activity of recombinant hEGF by Dot-ELISA method. Results： According to the result, the synthetic hEGF gene was expressed in transgenic tobacco plants and the recombinant protein could link to the antibody especially. Conclusion： It is an explanation that the Dot-ELISA method is simple and rapid, this method can be used to identify recombinant protein in plant transgenic engineering.