Abstract:

Methods The pmiRFP670-N1 plasmid and pET-28a plasmid were doubly digested with restriction endonucleases EcoR Ⅰ and Not Ⅰ to construct the pET-miRFP670 prokaryotic expression vector. The LyP-1 DNA sequence was introduced through point mutation to construct the recombinant expression vector pET-miRFP670-LyP1； the recombinant expression vector with correct sequence was transformed into the E.coli BL21 cells and the prokaryotic expression amounts of fusion proteins induced under different temperatures （16 ℃ and 37 ℃） and different concentrations of isopropyl-β-D-thiogalactopyranoside （IPTG）（0.1，0.5，and 1.0 mmol·L^－1） were detected by SDS-PAG electrophoresis； the fusion protein was purified by Ni-NTA resin affinity， and the prokaryotic expression amount of the miRFP670-LyP1 protein was detected； the endocytosis morphology of the miRFP670-LyP1 fusion protein in breast cancer 4T1 cells was observed under fluorescence microscope. Results The double digestion of recombinant plasmid showed that two DNA bands of about 5 343 and 973 bp were obtained， which was consistent with the sizes of the pET-28a vector and miRFP670 gene fragment. The DNA sequencing results showed that the LyP1 sequence was successfully inserted into the pET-miRFP670 expression vector. The soluble protein expression amount of miRFP670-LyP1 fusion protein was higher at 16 ℃ than that at 37 ℃. The miRFP670-LyP1 fusion protein with high purity was obtained by purification with Ni-NTA resin. The fluorescence imaging results showed that the miRFP670-LyP1 fusion protein could be efficiently endocytosed by the breast cancer 4T1 cells. Conclusion The prokaryotic expression vector pET-miRFP670-LyP1 is successfully constructed，the soluble protein expression amount of the fusion protein is higher at low temperature （16 ℃） than that at normal temperature （37 ℃）， and the fusion protein with high purity is obtained by affinity chromatography，and the fusion protein is efficiently endocytosed by the breast cancer 4T1 cells and displays near-infrared fluorescence. Objective To construct the prokaryotic expression vector of tumor homing peptide（THPs）-near-infrared fluorescent protein（NIRFP）-miRFP670 LyP1 fusion protein， and to purified fusion protein， and to investigate the near infrared fluorescence characteristics of the fusion protein.

Key words: Near-infrared fluorescent protein, Tumor homing peptide, Fusion protein, Prokaryotic expression, Endocytosis