The Expression Of The Human Antibacterial Peptide Ll-37 In Pichia Pastoris And It's Biological Properties

Since the discovery of penicillin,antibiotics have been the powerful weapon in the treatment of human infection of pathogenic microorganisms,but with long-term extensively use of the antibiotics,pathogens resistant increased seriously.It is an urgent need to develop new antibiotics,because it is more and more diffcult to find new antibiotics from microbes.The discovery of antibacterial peptides provides a broad source for the development of new antibiotics.With the resent researches,the ABP is a broad-spectrum antibiotic which can inhibit and kill resistant bacteria strains.Consequently,it is the most promising substitute for traditional antibiotics.Antibacterial peptides(ABP) include a series of small peptides with positive ions,most of which have abroad-spectrum antibiotic and they are significant ingredients during the course of congenital immunity in most creatures.Their function mechanism is different from antibiotics,so they don't cause bacterial resistance.There are three sources for ABP:isolating from natural resources,manual synthesis and expressing through gene engineering techniques.The first method of which is high cost,low productivity and boring production;the second method of which is very expensive,so neither of them can be used clinically.Due to the smallness of ABP gene,the acquisition of the gene is easy through manual synthesis ABP can be mass produced through micro-organism fermentation which have more practical meanings than the other ways.The purpose of this research is to develop recombinant ABP through the technique of gene engineering and protein engineering with the property right reserved and study the activity in vitro and in vivo;based on this slow release ABP have been synthesized.The researches of the study had laid the groundwork for the recombinant ABP manufacture production and safeguarding of animal husbandry.The study was unfolded through the following aspects.1 Using the preferential codon of Pichia pastoris,the gene of antibacterial peptide LL-37 of 141bp was synthesized manually according to the sequence from GenBank(GeneBank CAA86115).Especially a Kex2 signal cleavage site and glutamine were fused at respectfully 5' and 3' end of the antibacterial peptide gene.The antibacterial peptide gene was cloned into the expression vector pPICZα-A to construct the recombination expression vector pPICZα-A-LL-37.The recombinant vector was linearized by SacⅠand was electransformed into P.pastoris X-33 strain.Under the control of the promoter AOX1 (alcohol oxidase 1),the protein with a similar molecular weight to the native antibacterial peptides was expressed and secreted into supernatant.The antibacterial assay indicated that the peptide dispalyed strong antibacterial activites against Staphylococcus aureus(CowanⅠ), E.coli K99 and Salmonella pullorum.2 The requirement for the flask-shaking culture fermentation of the recombinant ABP Pichia pastoris was optimized.It was found that the recombinant LL-37 would be expressed with high level while the density of the yeast amounted to OD₆₀₀ value 5～6,the pH of the culturing BMMY medium was 6.0,the yeast was cultured under 28℃for 96 hours and 1% methanol was supplemented into the medium.The supematant with recombinant LL-37 exhibiting antibacterial activity against gram-positive and gram-negative bacteria. According to Agarose Diffusion Assay,the heat-stable and pH-stable characteristics of LL-37 peptide were examined.The pH-stable assay indicated that the peptide dispalyed the best activites while the pH was 5.0～6.0,and pH 2.0～12.0 still had certain activitys.The heat-stable assay indicated that the peptide could remain its inhibition activity after being treated for more than 3 hours in boiled water or autoclaving.The result of preservation period showed that:-20℃is the best preservation condition.Based on these characteristics, the recombinant antibacterial peptide display application foreground in the field of the prevention of disease and the additives of animal feeds tuff.3 Fourteen-day chicks were used,Preliminary study on the three formulations of antibacterial peptide LL-37 in chicken production performance and the impact of the protecting the chicks from the infection of E.coli O₁ was developed.MLD₁₀₀ of a strain of chick-origin E.coli O₁ was determined preliminary.Through 28 days of continuous water delivery Chicken,Chicken of weekly weight gain expected weight,the drug after the attack of mortality rates,and then compare their antibiotics in the above indicators of the differences.The results show that:after one week Medication,compared with the control group,(LL-37 + Cecropin P1) supernatant group weight gain increased significantly (P＜0.01);after two weeks Medication,the antibacterial peptide LL-37 three formulations group weight gain increased significantly(P＜0.01);medication for three weeks,four weeks, the chitosan microcapsules of the drug group weight gain Significantly increased(P＜0.05). After Medication one week,two weeks,three weeks and four weeks,the antibacterial peptide LL-37 three formulations group ratio of feed to gain decreased significantly (P＜0.01).After infected pathogenic E.coli O₁,antimicrobial peptide LL-37 three formulations group,ciprofloxacin group compared with the infection control group,the mortality rate decreased significantly(P＜0.01);antibacterial peptide LL-37 three formulations group prevention effective better than Ciprofloxacin group.4 Using twenty-one days chick,the effective of treating bacterial infection were studied. MLD₁₀₀ of a strain of E.coli O₁ to chick was determined preliminary.In the treating experiment,the chick with different dosage form recombinant antibacterial peptide LL-37 drinking was treated after 24h of infection with the dosage of MLD₁₀₀ of E.coli O₁.This treatment was lasted five days.The results showed that the recombinant antibacterial peptide LL-37 has preferable bioactivity in vivo.Especially,(LL-37+Cecropin P1) supernatant result is optimization and has the same treatment as Ciprofloxacin.The death rate of treatment with antibacterial peptide LL-37 and Ciprofloxacin degraded obviously(P＜0.05).However,the protection percentages and cure rates of antibacterial peptide LL-37 supernatant and Drug-loaded Chitosan are worse than Ciprofloxacin.