新型报导基因及绿色荧光蛋白发光特性的研究

Study of a Novel Report Gene and Green Fluorescent Protein

绿色荧光蛋白基因 (gfp)是一个极有潜力的新型报导基因。本文将突变型 gfp3′末端不同程度地截短 ,构建成表达载体 ,在大肠杆菌 BL 2 1中表达了不同长度的绿色荧光蛋白 (GFP) ,当其生色基因中 65位 Ser点突变成 Thr,则 GFP的激发波由 396nm增至 4 70 nm,不用 UV激发 ,仅在自然光源下就可看到强烈的绿色荧光 ,发光强度增加 6倍 ,若从 3′端截短 12个氨基酸密码 ,对发光无影响 ,但截短 75个氨基酸密码 ,则激发波长降到 360 nm,发光强度减少 12倍。实验证明GFP的发光机制 ,除与 65到 67位的三个氨基酸组成的生色基因有关外 ,与蛋白质分子的完整性 。

A novel report gene,green fluorescent protein gene(gfp),is developed now.Three expression vectors containing mutant gfp which were truncated from 3′ termine were constructed in the present paper.The mutation gfp with different length under the control by T 7 promoter were expressed in E. coli BL21 cells.The GFP S65T,in which the residue Ser had been altered to Thr at 65th position by dot mutation,has great advantages that the green fluorescent light can be directely observed under sun light because of moving exciting wave from 396nm to 470nm and increasing the light strength by five times.The intensity of luminescence of GFP S65TP with truncated length up to 12 amino acid residues from C terminus can not be changed,while truncated up to 75 amino acid residues from C terminus,the luminescence of GFP S65TH can not be observed.It is demonstrated that the mechanism of fluorophore formation is not only associated with chromophore consisting of three amino acids residues from 65~67 position but also with GFP structure conformation.