Cloning And Expression Of The Hybrid Antimicrobial Peptide Py-S? Gene In Pichia Pastris

Antimicrobial peptide is small molecular peptide produced by living organisms to protect against invasion of the surrounding pathogenic microorganism.It has significant inhibiting effects on bacteria,some fungi,viruses and tumor cells..Antimicrobial peptides are readily soluble in water,having good thermal stability and inhibitory activity in strong acid alkali conditions.And the above advantages determine their broad application prospect in food,agriculture and other fields.General access to antimicrobial peptides are: directly from biological separation,chemical synthesis and gene engineering of artificial expression.The antibacterial peptide synthesis uses genetic engineering technology,high activity,good physical and chemical properties,low production costs and High yield,so it is widely used.Hybrid antimicrobial peptides use genetic engineering technology,connect different antibacterial peptide fragments to form a new antibacterial peptide with stronger biological activity.Construction of hybrid antimicrobial peptides by changing the internal structure of natural antimicrobial peptides and the physical and chemical properties can overcome the shortcoming of natural antibacterial peptide,and thus integrating the advantages of two antibacterial peptides,is becoming a hotspot in current research.This experiment removed four cysteine of antimicrobial peptides PolyphemusinI and Spinigerin? with flexible chain links on the basis of Pichia pastoris preference codon Optimization translation Py and hybrid peptides Amino acid sequence,using the Primer 5.0 design primers by PCR reaction amplification of antibacterial peptide Py and hybrid peptide Py-S? gene.The amplified genes were cloned and identified and expressed in Pichia pastoris,the expression conditions were optimized,the biological activity and physicochemical properties of the expressed products were analyzed.Research indicates:The pPICZ?A-Py-S? hybrid antimicrobial peptide gene was successfully constructed.After extraction and by PCR amplification,recombinant plasmids pPICZ?A-Py and pPICZ?A-Py-S? were successfully integrated into the yeast genome.The recombinant yeast was induced to express on BMMY medium containing methanol.The expressed product to caonduct the SDS-PAGE electrophoresis after the SephadexG-25 chromatography.As the predict,has the 3.4 KD and 9.5KD single protein band,and the expected protein size consistent.According to the results of antimicrobial peptide expression,the optimum induction conditions were as follows: induction time 96 h,induction temperature 28 ?,culture medium initial pH value 6.0.Under the optimal conditions,the concentration of the expressed protein of antimicrobial peptide Py can reach 1.10 mg / mL.The induction time of porcine peptide Py-S? was 96 h,the induction temperature was 28 ?,and the initial pH value of culture medium was 6.5.Under the optimal conditions,the concentration of the expressed protein of antimicrobial peptide Py-S? was 1.25 mg / mL.Two antimicrobial peptides on E.coli,S.aureus,Salmonella and Shigella showed bacteriostatic effect.The effect on the E.coli is strong.The MIC of the two antimicrobial peptides to E.coli,S.aureus and Salmonella were 30 ug / mL,55 ug / mL,75 ug / mL and 20 ug / mL,35 ug / mL and 55 ug / mL.The antimicrobial effect of the hybrid antimicrobial peptide Py-S? was stronger than that of the antimicrobial peptide Py.In the stability study of antimicrobial peptides,two kinds of antimicrobial peptides were found to have good heat resistance,acid and alkali resistance and anti-radiation effect.Under the same treatment conditions,the antibacterial effect of the hybrid peptide was always higher than that of the antimicrobial peptide Py.After recombinant yeast passaging 10 times and PCR amplification confirmed that there is no loss of the target gene,the expression of protein is still antibacterial effect.In summary,the study successfully built the eukaryotic expression vector pPICZ?A-Py and pPICZ?A-Py-S?,and induced expression in Pichia pastoris.The results showed that the expressed products had certain bioactivity,the antibacterial effect of the hybrid antimicrobial peptides was improved,the studying of Peptide lay a certain foundation for further study.