Prokaryotic Expression Of Recombinant Rat Lysozyme N-terminal Fragment And Its AGEs Binding Activity

Objectives: To highly express the recombinant rat lysozyme N-terminal fragmentin prokaryotes and to investigate the AGEs-binding activity of the recombinant fragment in vitro.Methods: ①The total RNA was extracted from leucocytes of Sprague-Dawley rats,and the cDNA sequences for rat lysozyme and its N-terminal fragment were prepared using RT-PCR techniques, respectively. ②The rat lysozyme gene was inserted into a vector pMD18-T to construct the recombinant cloning vector, pMD18-T/rLyz, for amplification. Double-restriction endonuclease digestion and gene sequencing were carried out to confirm the right DNA sequence. ③The sequence, encoding for the N-terminal fragment of rat lysozyme, was then inserted into an expression vector, pET-30α(+), to construct the recombinant expression vector, pLY77, which was identified by double restriction endonuclease digestion and sequencing ④ The recombinant expression vector pLY77 was then transfected into E. coli Rosetta(DE3) and BL21(DE3) for expression of the recombinant rat lysozyme N-terminal fragment through induction by IPTG. Optimization of the induction conditions including temperature, IPTG concentration and induction time was done to obtain the maximum yield of the recombinant fragment. ⑤The expression products were purified by using Ni~+-NTA agarose chromatography, and the prepared rat lysozyme fragment LY77 was identified as a highly pure peptide by using SDS-PAGE and Western blotting techniques. ⑥Advanced glycation end products (BSA-AGEs) were prepared in vitro, and it was demonstrated that the recombinant peptide LY77 exhibited a potent binding activity with lab-prepared bovine serum albumin-AGEs in vitro.Results: 1) The recombinant vector was successfully constructed in the study: ① The total RNA isolated gave three typical bands in agarose electrophoresis: 28s, 18s...