Study On The Activity Of The Antibacterial Peptide Cecropin P1 Expressed In Pichia Pastoris

Antibiotics,which has been playing a very important role either in the field of animal husbandry or human medicine,is the powerful weapon in the prevention against bacterial infection and the treatment of domestic animal diseases.Besides, antibiotics can also promote the growth of animals as feed additive.However the abuse of antibiotics also brings some serious problems:drug remnant and the emergence of antibiotic-resistant bacterium which is much rapider than the development of new antibiotics.Therefore,"No Poison,No Remnant,No Drug Resistance"is the request of a new genera tion of antibacterial candidate.Antibacterial peptides(ABP) is a kind of peptide that is created by the immunological system of organism to defending the infection of pathogens.It is the important component of innate immunological system.Antibacterial peptides will not induce the resistance of bacteria because of its unique antibacterial machnism,which suggests that ABP can be a potential substitute of traditional antibiotics.There are three sources for ABP:isolating from natural resources,manual synthesis and expressing through gene engineering techniques.Isolating from natural resources costs heavily and has low productivity with boring procedures.Manual synthesis is very expensive and the preciseness will have negative correlation with the number of amino acid.Gene engineering techniques are prefered to obtain ABP. The purpose of this research is to develop recombinant pig intestine antibacterial pep tide cecropin P1 through the technique of gene engineering and protein engineering and to study the in vitro and in vivo activity,which lay a foundation for the mass production of ABP and the safeguarding of animal husbandry.The study was conducted from following aspects:1 Using the preferential condon of Pichia pastoris,the gene of antibacterial peptide cecropin P1 of 117bp was synthesized manually according to the sequence from GenBank(Gene Bank P14661).Especially a Kex2 signal cleavage site and glutamine were fused in respectfully 5' and 3' end of the antibacterial peptide genes.The antibacterial peptide genes was cloned into the expression vector pPICZa-A construct the recombineant expression vectors pPICZa-A-cecP1.The recombinant vector was linearized by Sac I and electransformed into Pichia pastoris SMD1168 strain by electransformation.Under the control of the promoter AOX1 (alcohol oxidase 1),the proteins with a similar molecular weight to native antibacterial peptides was expressed and fused into supernatant.The antibacterial assay indicated that the peptide dispalyed strong antibacterial activites against Escherichiacoil K99,Staphylococcus aureus, Salmonella gallia-rum and Bacillus subtilis.2 There is only the protease B activity left in SMD1168 with Protease A and carboxy Y left,which is benefitial to the stability of recombinant antibacterial peptides in the expressed supernatant.Therefore SMD1168 strain is suitable for the expression of the low molecular biological peptide comparing with X-33 strain and GS115 strain.The requirements for the flask-shaking culture fermentation of the recombinant ABP Pichia pastoris was optimized.It was found that the recombinant cecropin P1 would be expressed with high level while the density of the yeast amounted to OD₆₀₀ value 7-8, the pH of the culturing BMMY medium was 6.0, the yeast was cultured under 28℃for 72 hoursand 0.5% methanol was supplement ed into the medium.The supernatant with recombinant cecropin P1 exhibiting antibacterial activity against gram-positive and gram-negative bacteria,and there was some correlationship between dosage of agent and antibacterial activity.According to Agarose Diffusion Assay,the heat-stable and pH-stable characteristics of cecropin P1 peptide were examined.3 Kunming mouse were used to study the elementary effectiveness of preventing and treating bacterial infection.In the preliminary experiment,the MLD₁₀₀ of Staphyoc occus aureus (Cowan I) and Escherichia coil K99 to mouse were determined.The mice with celiac injection of 240μg purified recombinant antibacterial peptide cecropin P1 were detected alive in 48 hours after infection with 10 folds dosage of MLD₁₀₀ of Staphylococcus aureus (Cowan I) and Escherichia coil K99.The protection percentage of two bacteria were respective 87.5% and 75%. In the treating experiment,the mice with celiac injection of 240μg purified recombinant antibacterial peptide cecropin P1 had respectively got 75% and 62.5% protection after infection with the dosage of MLD₁₀₀ of Staphylococcus aureus (Cowan I) and Escherichia coil K99.These results indicated that the recombinant antibacterial peptide cecropin P1 has preferable in vivo bioactivity.