The Design And Synthetic Of The Hybrid Antimicrobial Peptide Lf-Ap Gene And Expression In Pichia Pastoris

Hybrid antimicrobial peptide is a new class of antimicrobial peptides,which can use genetic means to connect two or more different antibacterial peptide fragments together.Thereby, we can form a new model of antimicrobial peptides,which have more antibacterial peptide activity. A novel hybrid antimicrobial peptide is based on the optimization and reformation of natural antimicrobial peptides.At the same time,we also need to optimize the structure-activity relationship and physicochemical properties for overcoming the shortcomings of natural antimicrobial peptides.This experiment simulates the structure of hybrid antibacterial peptide Lf-Ap through bioinformatics software and chooses the preferred codon of Pichia pastoris to design and synthesize primer and optimize the designing of hybrid antibacterial peptide Lf-Ap gene sequences.At last we successfully amplify the hybrid antibacterial peptide gene about 98bp--Lf-Ap by the PCR technology and complete the construction of vector.Recombinant plasmid was lineared by single enzyme and was transformed into host bacterium GS115 by electroporation. And we success of the screening of medium high copy transformation strains by selective containing Zeocin. The positive strains of yeast genome extraction verified by PCR. The purpose gene has been successfully verified by observation of gel imaging system.The correct positive GS115- Lf-Ap was induced by methanol in BMMY medium. And expression products were analysied examined by electrophoresis of SDS-PAGE. So as we can judge that the size of the expression products meets the forecasting or not.Through the method of agar hole diffusion, on E.coli, Staphylococcus aureus, Salmonella enteritidis,Niger and beer yeast as sensitive strains, verifies the bacteriostatic activity.The results show that hybrid antimicrobial peptide Lf-Ap has better antimicrobial activity on E.coli, staphylococcus aureus, salmonella.But it has no effect on aspergillus Niger and beer yeast. Genetic stability, after passaging 50 times, the target gene is not lost, and the expression the product still has good antibacterial activity.To sum up, this study successfully cloned the hybrid antimicrobial peptide Sα-Jp gene, and expressied in eukaryotic expression system. Under the optimized expression condition, obtained the highly effective expression protein. Laid a good foundation for the research about the new hybrid antimicrobial peptides.