Expression,Purification And Biological Activity Detection Of Melittin

Natural antibacterial peptides come from a wide range of sources,not only from animals and plants,but also from insects.Bee venom peptides have gradually attracted wide attention from scientists at home and abroad due to their broad-spectrum and high-efficiency antibacterial activity and low drug resistance.The current schemes for obtaining bee venom peptides include chemical synthesis,crude extraction of bee venom dry powder and genetic engineering methods.The chemical synthesis of bee venom peptides is costly and takes a lot of time.There are many steps to extract bee venom peptides from bee venom dry powder,with low yield and low purity,and it is difficult to meet scientific research and clinical needs.The production of bee venom peptides by genetic engineering expression strategy has many advantages such as high yield,short cycle,low cost and easy purification.Therefore,it is an effective method to produce bee venom peptides in large quantities by genetic engineering.In this study,the SUMO-Melittin gene was synthesized and constructed into the p ET3c expression vector,and the positive recombinant expression plasmid p ET3c-SUMO-Melittin was selected.The recombinant plasmid was transformed into E.coli BL21?DE3?competent cells,and stable and highly efficient expression was obtained.It has been verified that the engineering bacteria have good passage stability and expression stability.Optimize its expression conditions,and carry out large-scale shake flask culture according to the best expression conditions.After purification by Ni affinity chromatography,high-purity fusion protein was obtained.After Western blot identification,it was digested with SUMO and purified by Ni affinity chromatography to obtain high-purity recombinant bee venom peptides.The antibacterial activity,hemolytic activity,cytotoxicity and antitumor activity of the recombinant bee venom peptides were tested.The main experimental results are as follows:?1?The recombinant expression plasmid p ET3c-SUMO-Melittin was successfully constructed and expressed in Escherichia coli.The optimal induction time was when the bacterial solution was OD₆₀₀=0.8,and the final IPTG concentration was 0.5 m M,and induction was conducted at 37?for 5 h.?2?After purification by Ni affinity chromatography,the SUMO-Melittin fusion protein was obtained with a size of 21 k Da.After digestion with SUMO,purification by Ni affinity chromatography was performed again to obtain Melittin protein,which was analyzed by SDS-PAGE and mass spectrometry.And HPLC analysis,the molecular weight is 2.84 k Da,and the purity is greater than 95%.?3?The purified melittin had an MIC of 16,8,8 against E.coli ATCC 25922,Salmonella typhimurium CMCC 50115,Shigella flexneri ATCC 12022,Klebsiella pneumoniae CMCC10031,Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 63501,respectively 16,32,2,32?g/m L.Recombinant Melittin at a concentration of 3.18?g/m L can lyse 5%human red blood cells.Recombinant Melittin at a concentration of 2.94?g/m L,3.49?g/m L and 4.42?g/m L can kill 50%of RAW 264.7,HEK 293 cells and NIH3T3 in 24 hours cell.Recombinant Melittin at a concentration of 4.36?g/m L and 5.18?g/m L can kill 50%of Hela cells and HepG2 cells in 24 hours.The purified melittin had an MIC of 16,8,8 against E.coli ATCC25922,Salmonella typhimurium CMCC 50115,Shigella flexneri ATCC 12022,Klebsiella pneumoniae CMCC 10031,Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC63501,respectively 16,32,2,32?g/m L.Recombinant Melittin at a concentration of 3.18?g/m L can lyse 5%human red blood cells.Recombinant Melittin at a concentration of 2.94?g/m L,3.49?g/m L and 4.42?g/m L can kill 50%of RAW 264.7,HEK 293 and NIH3T3 in 24 hours cell.Recombinant Melittin at a concentration of 4.36?g/m L and 5.18?g/m L can kill 50%of Hela and HepG2 cells in 24 hours.