Study on Gene Prokaryotic Expression of Ginkgo biloba Antimicrobial Peptide Protein and Antimicrobial Activity of Its Re- combinant Protein in vivo and in vitro / 中国药房

ABSTRACT

OBJECTIVE： To obtain Ginkgo biloba antimicrobial peptide （GBA） recombinant protein， and to investigate in vivo/in vitro antimicrobial activity of the protein so as to provide experimental basis for solving bacterial resistance and large-scale production of new plant-derived antimicrobial agents. METHODS： Based on gene technology， according to GBA gene sequence   （FJ 865399） published by Genebank， recombinant expression vector plasmid pET32a（+）-GBA was constructed. Prokaryotic expression of recombinant protein was conducted by Escherichia coli， and then the protein was purified and identified by gel electrophoresis and Western blotting. Drug sensitivity of obtained recombinant protein to E. coli， Staphylococcus aureus， Pseudomonas aeruginosa， Salmonella typhimurium were investigated by Kirby-Bauer test. The minimum antibacterial concentration （MIC） and minimum bactericidal concentration （MBC） were determined by broth dilution method. The protective effects of recombinant protein on S. aureus infection model mice were investigated. RESULTS： Target recombinant protein was expressed successfully and purified （molecular weight of 32 kDa）. The recombinant protein was moderately sensitive to S. aureus and low sensitive to other three bacterias. MIC and MBC of the recombinant protein to S. aureus were （50.00±5.00）mg/mL and（138.33±12.58）mg/mL， and MIC was significantly higher than those to other 3 kinds of bacterias （P＜0.05）. High-dose of recombinant protein （8.0 g/kg） could significantly reduce the S. aureus-induced mortality of mice （P＜0.05）， and had similar protective effect as positive drug penicillin. CONCLUSIONS： Obtained recombinant protein has obvious antimicrobial effects on S. aureus， inhibits E. coli and P. aeruginosa to certain extent and shows poor inhibitive effect on S. typhimurium. High-dose of  recombinant protein shows significant protective effect for S. aureus infection model mice.