New Antibactierial Peptides AWRK6 Optimizat Construction And Expression In Pichia Pastoris

Peptide (antibacterial peptides, ABP), also known as antimicrobial peptides (antimicrobial peptides, AMP) or peptide antibiotics (peptide antibiotics), defending exogenous pathogens and malignant cells, is a class of small molecule active peptides that is produced by the non-specific response in immune defense system in general. Antibacterial peptide is a small molecule peptide generally containing 10-50 amino acids, with the features of broad-spectrum antibacterial activity, strong thermal stability, small molecular weight and low immunogenicity, becauese of bactericidal mechanism unique and easy to produce drug-resistant, it is expected to develop a new generation of peptide antibiotics.Research on antibacterial peptide has lasted for several ten years and some peptide antibiotics are in the phase of clinical trial in abroad. It is in the initial stage of research on antibacterial peptide of Rana dybowskii in molecular level and just several sequences was reported so far. Natural antimicrobial peptides not only have many other antibiotics and incomparable advantages, but also has its inadequate. Some natural antimicrobial peptides have both haemolyticus and sterilizing, others antimicrobial peptides is biotoxicity by itself, it has not applied in clinical treatment. So the reform of natural antimicrobial peptides is a new direction at present.At present the molecular design scheme of antibacterial peptides reform is mainly to analyze the biochemical properties of the natural antimicrobial peptides, according to factors known to influence activity, to design of new antimicrobial peptides. Or by comparing the structure of the known antimicrobial peptides, to identify conserved sequences, and screen new antibacterial peptides with excellent antimicrobial activity.Dybowskin-2CDYa(SAVGRHGRRFGLRKHRKH)is a new natural antibacterial peptide which is found and named by our laboratory in frog skin, rich in Arg. Bioinformatics analysis showed that the instability coefficient (Instability index) reached 52.19, but proteins that the coefficient is less than 40 can exist stably instability in theory. According to the existing literature, reform antibacterial peptides dybowskin - 2CDYa by means of replacing all Arg by the Lys, and replacing Gla in the second place by Trp, and then antimicrobial peptides after transforming called AWRK6. AWRK6 (SWVGKHGKKFGLKKHKKH) is composed of 18 amino acids, molecular weight is 2130.5, to predict the secondary structure by the method of HNN and find out its structure, it is mainlyα-helix, isoelectric point is 9.60 and instability coefficient is -6.74, the result indicate antimicrobial peptide AWRK6 is more stable than dybowskin-2CDYa. The AWRK6 gene after transforming get full-length genes by means of SOE-PCR. Connect the yeast secretory expression vector pPICZα-A, and construct pPICZα-AWRK6 yeast expression plasmid. Recombinant plasmid is digested with SacI, and electroporatinto Pichia pastoris X33, then construct eukaryotic expression system. Use Zeocin to select positive strains, and prove positive strains by PCR. Positive transformants were induced by methanol, useα-factor primer, 3’AOX primers to amplify cDNA of reverse transcription to obtain amplification products, it is about 300 bp. It is consistent with the size of the target gene AWRK6, indicating that the gene has been inserted into the host genome and transcription. Measure the activity of fermentation liquid, and antibacterial activity showed that the recombinant antibacterial peptide AWRK6 has inhibitory activity against Gram-negative bacteria (Escherichia coli O157), and Gram-positive bacteria (Staphylococcus aureus).Conclusion: Instead all of the arginine residues of the antimicrobial peptide to lysine residues, it could improve their antimicrobial activity. New antimicrobial peptides AWRK6 genes successfully is expressed in Pasteur pichia X33 expression system. The secondary structure of antimicrobial peptide is mainlyα-helix amphiphilic spiral structure. AWRK6 has antibacterial activity to Escherichia coli and staphylococcus aureus.