恒波长同步荧光法测定吐温80-硫酸铵液固萃取体系中绿色荧光蛋白标记的融合蛋白  

Determination of Fusion Protein Tagged Green Fluorescent Protein in Tween80-Ammonium Sulphate Liquid-Solid Extraction System by Constant-Wavelength Synchronous Fluorescence

作　　者：沈静茹[1] 靳光才[1] 余学红[2] 韦康[1] 林敏[1] 

机构地区：[1]中南民族大学化学与材料科学学院国家民委分析化学重点实验室,武汉430074 [2]中南民族大学校医院,武汉430074

出　　处：《中南民族大学学报（自然科学版）》2010年第1期22-27,共6页Journal of South-Central University for Nationalities：Natural Science Edition

基　　金：国家民委重点基金资助项目(29475219)

摘　　要：培养了含六聚组氨酸绿色荧光蛋白修饰融合蛋白的工程菌,对从工程菌中收集的蛋白原液用普通荧光和恒波长同步荧光2种荧光表征方式进行了比较,探究了分离蛋白的液固萃取体系中各因素(如硫酸铵浓度、吐温80浓度、酸度)对蛋白质荧光特性的影响,确定了萃取体系中适用的恒波长同步荧光测定法.运用该法得到液相的工作曲线线性相关系数达0.999 35,固相工作曲线线性相关系数为0.997 48,原始吐温80-硫酸铵液固萃取体系分离菌中目标蛋白,一次萃取固相收得率可达41.31%,调节pH 6.2时一次萃取固相收得率可达63.24%,进一步提高选择性,加入聚乙二醇修饰物可提高至93.50%.The engineering bacterium consisting of six-polyhistidine and green fluorescent protein （GFP） was cultivated. The general fluorescence and constant-wavelength synchronous fluorescence for the characterization of the protein obtained from the engineering bacterium were compared. The various factors involved in the liquidsolid extraction system such as the ammonium sulphate concentration, Tween 80 concentration and acidity on the characteristics of the fluourescence were explored. The method of constant-wavelength synchronous fluorescence was confirmed. According to this method, the linear correlation coefficient of the liquid phase is 0. 999 35 and that for the solid phase is 0. 997 48. Using the Tween 80- ammonium sulphate liquid -solid extraction system the first solid phase yield of the target protein is 41.31%. The yield increased to 63.24 % at pH 6.2. With addition of polyethylene glycol modifier, the first solid phase yield of the target protein was further improved to 93.50%.

关 键 词：蛋白分离 纯化 液-固萃取 恒波长同步荧光 

分 类 号：O657.31[理学—分析化学]