Preparation And Activity Analysis Of Recombinant Fusion Protein Of Streptavidin And Green Fluorescent Protein

Due to the extremely strong affinity between streptavidin(SA)and biotin,the SA-biotin system exhibits a powerful function in immunodetection signal amplification.Therefore,the development of molecular detection technology based on the SA-biotin system,the application prospects of which will be quite broad.In this thesis,SA gene and enhanced green fluorescent protein(EGFP)gene were used to construct recombinant fusion proteins SA-EGFP and c SA-EGFP.It replaces the labelled secondary antibody by detecting biotin-labelled primary antibody to improve the sensitivity,reproducibility and reduce the cost of immune detection.The main results are as follows:(1)The prokaryotic expression vector p ET-28 a used to be modified to obtain a prokaryotic expression vector containing only one 6×His tag sequence downstream of the multiple cloning site(the modified vector used to be named p ET-28n).(2)Escherichia coli BL21(DE3)and Rosetta(DE3)have been used for expression of SA-EGFP,and SDS-PAGE results confirmed that no specific band regarded close to the theoretical molecular weight(45.3 k Da),Western Blotting results confirmed that a faint spot regarded close to 30 k Da.Escherichia coli BL21(DE3)and Rosetta(DE3)have been used for expression of his-SA-EGFP,and SDS-PAGE results confirmed that no specific band regarded close to the theoretical molecular weight(45.3 k Da),Western Blotting analysis discovered that a specific band seemed close to the theoretical molecular weight(45.3 k Da),however its expression degree used to be low and close to 30 k Da weak spots additionally appeared.(3)Pichia pastoris GS115 was used for expression of SA-EGFP,and the results of Western Blotting confirmed that no specific band seemed close to the theoretical molecular weight,however spots regarded close to 30 k Da.(4)Escherichia coli BL21(DE3)was used for expression of c SA-EGFP,and SDS-PAGE analysis confirmed that a specific band appeared close to the theoretical molecular weight,and the protein was the most expressed at 28 ℃ and 0.4 m M IPTG for 8 hours;Western Blotting results confirmed that it appeared close to the theoretical molecular weight,and specific band.(5)Ni-NTA resin was used to purify the induced-expressed fusion protein c SA-EGFP.SDS-PAGE results confirmed that the purified protein band was close to the theoretical molecular weight,and the band was single.(6)The fluorescence intensity of the fusion protein c SA-EGFP increased with the amplify of its amount,and the fluorescence used to be nevertheless determined even when the fusion protein c SA-EGFP was only 25 ng.Moreover,molecular hybridization using Biotin-BSA and fusion protein c SA-EGFP,the fluorescence intensity improved with the amplify of Biotin-BSA amount,and when Biotin-BSA used to be 5 pg,vulnerable fluorescence could still be observed.The fusion protein c SA-EGFP prepared via the above series of research had excellent biological activity and could be applied to the SA-biotin system based immunodetection assay system.