甲胎蛋白-增强型绿色荧光蛋白重组体的构建表达及其测量特性分析  

The construction and expression of AFP-enhanced green fluorescent protein recombinant and the analysis of measurement characteristics

作　　者：张健 葛丹红 王雪亮 

机构地区：[1]上海市临床检验中心,上海200126

出　　处：《检验医学》2014年第6期659-663,共5页Laboratory Medicine

基　　金：上海市科委自然科学基金资助项目(09ZR1427300)

摘　　要：目的构建增强型绿色荧光蛋白(eGFP)标记的甲胎蛋白(AFP)的融合蛋白,并对其稳定性和测量特性进行分析。方法从pcDNA3.0质粒中扩增eGFP序列,插入质粒PET28a,构建eGFP表达质粒PET28a-eGFP。根据美国国立生物技术信息中心(NCBI)数据库中的AFP编码序列,应用序列合成方法合成AFP编码序列,并加入连接eGFP的铰链序列,与eGFP序列链接,构建表达质粒PET28a-eGFP-AFP。分别表达和纯化重组蛋白eGFP和eGFP-AFP,分析重组蛋白的荧光特性及稳定性。结果成功构建和纯化重组蛋白eGFP和eGFP-AFP,荧光光谱分析显示其激发和发射光谱一致。最佳激发波长和发射波长分别为450和509 nm,且荧光可以稳定12个月。表达的eGFP-AFP蛋白可以与常规测定AFP的免疫试剂发生反应。结论构建表达的重组蛋白eGFP-AFP的荧光特性与eGFP蛋白一致,没有光谱特性变化,并可与常规AFP测定试剂发生反应,为研究eGFP标记的AFP用于校准品和质控品的研究奠定基础。Objective To construct the fusion protein of alpha fetoprotein （AFP ）-enhanced green fluorescent protein（eGFP）recombinant,and to analyze the stability and measurement characteristics.Methods The sequence of eGFP was amplified from pcDNA3.0 plasmid and inserted PET28a plasmid to construct expression plasmid PET28a-eGFP.The coding sequence of AFP was synthetized according to the coding sequence of AFP from the National Center for Biotechnology Information （NCBI）database,added and linked to eGFP sequence by a linker sequence to construct expression plasmid PET28a-eGFP-AFP.Recombinant protein eGFP and eGFP-AFP were purified,and the measurement characteristics and stability were analyzed.Results The expression plasmid of recombinant protein eGFP and eGFP-AFP were constructed,and the proteins were purified.Recombinant protein eGFP and eGFP-AFP had the same excitation spectrum and emission spectrum,which were 450 and 509 nm optimally,and its fluorescence could be stable over 1 2 months.eGFP-AFP could be tested for the level of AFP by routine AFP immunoassay.Conclusions Recombinant protein eGFP and eGFP-AFP have the same fluorescence characteristics and could react with routine immunoassay which establishes the foundation for the next research using eGFP-AFP as calibration and quality control materials.

关 键 词：甲胎蛋白 绿色荧光蛋白 荧光光谱 融合蛋白 校准品 

分 类 号：R446.1[医药卫生—诊断学]