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

摘要

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.%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。