Abstract: 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 12 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.

Key words: Alpha fetoprotein, Green fluorescent protein, Fluorescence spectrum, Fusion protein, Calibration