Construction And Application Of Expression Vector With Multi-opy Antibacterial Peptide Gene Tandem In The Same Direction

Now days antibiotics are widely used in medical and feed industry，but thetraditional use of antibiotics leads to more and more drug resistant strains because thedrug resistance of bacteria growing. Effect of antibiotics for clinical anti-infectiontreatment has been challenged. At the same time, along with the demand of people intheir own health, food quality and environmental quality is becoming more and morehigher, people become more and more alerter in the side effects of antibiotics, forexample, antibiotics in the feed industry has faced elimination and disable. Therefore,antibiotics will be replaced by products what is conducive to human and animal health,environmental protection and improving the economic benefit.Antibacterial peptide as a kind of heat stability and broad spectrum antibacterialactivity of peptides widely exists in insects, animals, plants and human body, and is animportant part of the host immune defense system. Antibacterial peptides not onlyhave efficient broad-spectrum killing ability against gram negative bacteria and grampositive bacteria but also have inhibitory effect for some viruses, fungi and protozoa[1].In addition, the antibacterial peptides have selective destruction effect on some tumorcells, but have no obvious side effects on mammals and insects, the particularmechanism of action of antimicrobial peptides produce few bacteria drug resistancemutations as a drug treatment. Therefore, Antibacterial peptide has wide applicationprospect in the human production and living in the future.The level of antibacterial peptides in the biological body is low and theseparation is difficult，and the cost of chemical synthesis method is too high. Most ofthe antimicrobial peptide’s molecular weight is small, after building a single copygene recombinant plasmid use conventional genetic engineering methods, objectivegene expression in the host cell is low, unstable and easy to be degraded by the hostprotease; In addition, the antibacterial peptide itself has a role of prokaryotic hostbacteria, and is not suitable for direct expression in prokaryotic expression system.These problems result in the expression of antimicrobial peptide to be efficient[2].Gene tandem technology connect small molecule peptide series in the fore andaft direction，then increase gene copy number and the molecular weight. Gene tandemtechnology overcomes the low amount of small molecular peptide in vitro expression. Pasteur pichia expression system is a kind of mature and efficient eukaryoticexpression system, It not only has breed quickly prokaryotic expression system, thecharacteristics of low cost and convenient operation, but also has the advantages ofeukaryotic expression system to express protein’s correct processing, folding andmoderate glycosylation.This experiment builds multi-copy tandem in the same direction of antibacterialpeptide gene using genetic series technology, and chooses the true pichia expressionplasmid as plasmid carrier, successful gains yeast expression plasmid containingmulti-copy antibacterial peptide gene tandem in the same direction, lay the foundationfor further stability efficient expression in pichia expression system of the targetprotein.