Expression Of Three Antimicrobial Peptides Gene In Pichia Pastoris And Saccharomyces Cerevisiae

Human health suffers a serious threat due to long-term and extensive use of antibiotics which cause pathogenic bacteria drug resistance problems and its residue problems in animals. Therefore, developing a highly efficient and safe alternatives to antibiotics has become an important direction to feed additive research. In this moment, antimicrobial peptides with a variety of biological functions shows a wide application potential.Is CT is a kind of linear and cytotoxicity peptide which derived from Madagascar black claw(Opisthacanthus madagascariensis). It is composed of 13 amino acid residues with alpha helix structure and its molecular size is 1502.9Da without cell selectively. Study shows Is CT derivatives [L6,K11]-Is CT has better antimicrobial activity than Is CT, and [L6,K11]-Is CT does not work on red blood of mammlas with no hemolysis activity; Clavanin from handle sea squirts(Styela clava) blood cells is composed of 23 amino acid residues with a alpha helix structure. It is similar to Magainins(separated from Xenopus laevis) on molecular size, structure and antimicrobial activity; Pa-AMP05 separated form pokeweed(Phytolacca americanan L.) seeds cconstitutes of 38 amino acid reduse. It has six cysteine, compose three pairs of disulfide bond formation. The molecular of Pa-AMP05 is 4193.8Da,and its isoelectric point is 9.3, alkaline.This study is going to use genetic engineering technology to expression Is CT derivatives, Clavanin and Pa-AMP05 in Pichia pastoris and Saccharomyces cerevisiae respectively. It is established the theory and the practice foundation for the production and researchment of antimicrobial peptides Is CT derivatives, Clavanin and Pa-AMP05 and other antimicrobial peptides. Following is the main content of this study: 1) According to codes preferences of Pichia pastoris, adopting overlap region geneamplification splicing method, design and synthetize Is CT derivatives, Clavanin andPa-AMP05 genes.2) Constract Pichia expression vector of Is CT derivatives, Clavanin and Pa-AMP05respecively. After transformation of Picha pastoris and induce fermentation of shake flask,detect antimicrobial activity of fermented supernatant. The results show the threerecombinant Picha have antimicrobial activity against S. aureus ATCC 25923 and E. coliATCC 8739. And strain X33/p PICZαA-Is CT has largest bacteriostatic ring diameter aferinducing 72 h, and in that montent,this strain has the highest yield of protein expression;strain X33/p PICZαA-Clavanin has largest bacteriostatic ring diameter afer inducing 72 h,and in that montent,this strain has the highest yield of protein expression; strainX33/p PICZαA-AMP05 has largest bacteriostatic ring diameter afer inducing 96 h, and inthat montent,this strain has the highest yield of protein expression. 3) Constract Saccharomyces cerevisiae expression vector of Is CT derivatives, Clavanin andPa-AMP05 respecively. After transformation of Saccharomyces cerevisiae and inducefermentation of shake flask, detect antimicrobial activity of fermented supernatant. Theresults show the three recombinant yeast have antimicrobial activity against S. aureusATCC 25923 and E. coli ATCC 8739. And strain INVSc1/p YES2α-Is CT has largestbacteriostatic ring diameter afer inducing 72 h, and in that montent,this strain has thehighest yield of protein expression; strain INVSc1/p YES2α-Clavanin has largestbacteriostatic ring diameter afer inducing 72 h, and in that montent,this strain has thehighest yield of protein expression; strain INVSc1/p YES2α-AMP05 has largestbacteriostatic ring diameter afer inducing 96 h, and in that montent,this strain has thehighest yield of protein expression.