Coloning And Expression Of Antibacterial Peptide DYBOWSKIN-2CDYA And It's Application In Poultry Feed

Rana dybowskii is one kind of amphibian which mainly distribute in northeast of China,and it's skin surface mucus is rich of antimicrobial substance which called antimicrobial peptides.The amphibian skin antimicrobial peptides are the first line of defense in innate immunity,which is a kind of small peptides usually constituded by about 10 to 40 amino acid residues secreted by skin granular glands,and there is usually a positive charge on it's surface.Antimicrobial peptides have broad-spectrum antibacterial,antiviral,antitumor,antiprotozoal and other biological functions,and also have lots of characteristics such as thermal stability,low molecular weight and small immunogenicity.Antimicrobial peptides has become a promising new generation of antimicrobial peptide antibiotics and food additives,feed additives based on its unique bactericidal mechanism and low drug resistance.Currently there are a variety of peptide on the stage of clinicaltest abroad.Research on Rana dybowski skin antimicrobial peptides on molecular level is at an initial stage,Therefore,a research on gene structure,physiological and biochemical properties,peptide chain structure optimization,function and industrial production and other aspects of Rana dybowski skin antimicrobial peptides has important theoretical and practical significance for its application and development.In this study,degenerate primers were designed based on 5' conservative sequences of variety of Rana antibacterial peptide gene and used to amplify the cDNA library which reverse-transcripted by the total RNA extracted from the skin of Rana dybowskii.Then the gene library of antibacterial peptide got from Rana dybowskii was sequenced randomly chosen.The sequenced antibacterial peptides were alignmented by CLUSTALW.The results showed that there are 11 kinds of antimicrobial peptides we got from Rana dybowskii.The dybowskin-5,6,7,8,9 belonging to the Temporin family and dybowskin-1,2,3 belonging to Brevinin-1 family.The dybowskin-4 is similar to the Japonicin-1 seperated from Rana japonica with I residue difference only.Both dybowskin-10 and 11 are a new family series which have little similarity to other peptide families reported previously.These two peptides our laboratory first discovered were named:Dybowskin-2CDYa(sequence:SAVGRHGRRFGLRKHRKH),Dybowskin-2CDYb(sequence:SAVGRHSRR FGLRKHRKH).Predicted by EXPASY biological software,he physical and chemical properties of Dybowskin-2CDYa and Dybowskin-2CDYb were different from other antimicrobial peptides significantly:more positive charge;no amidation modification of C terminal;higher pI,strong alkaline and hydrophilic.The pPICZ?/Dybowskin-2CDYa expression vectors constructed with Dybowskin-2CDYa gene secreted Pichia expression vector pPICZawas sequenced and the result showed that the recombinant plasmid reading framewas in accordance with our design.Then the expression plasmid was transformed into Pichia pastoris X33 conducted induced by methanol.The expression production was precipitated in TCA and analyzed with the 15%Tricine-SDS-PAGE,the band of 2 kDa was got.The fermentation supernatant was seperated by reverse phase chromatography,and then the primary chromatographic elution peaks was collected,frozen-dried,and then have the antibacterial activity test.The results showed a retention time of 21.78 min elution peaks have antibacterial activity,which was account for about 6.32%to the total peak area.Protein content of the fermentation supernatant was 4.79 ?g/?L.The theoretical expressionquantity of fussion peptide caculated by area normalization method was about 0.30 g/L.More peptide was purified by using semi-preparative reverse column(10 x 150 mm,5 ?m,ODS)from fermentation broth,and the antibacterial activity of purified Dybowskin-2CDYa was detected by Agar diffusion assay.The results showed that:Dybowskin-2CDYa bacteriostatic efficacy on Bacillus cereus,Escherichia coli 0157,hemolytic Acinetobacter baumannii and Enterobacter aerogenes.Natural antimicrobial peptides have many advantages over other antibiotics but also has its defective such as hemolytic and toxicbiological property,therefore the clinical and other application is limited.The instability coefficient of Dybowskin-2CDYa reached 52.19,which suggests that the protein is unstable,coefficient of less than 40 is considered able to exist stably.The recombinant yeast Dybowskin-2CDYa will lose it's antibacterial activity after heating 5min in 100? which shows its low thermal stability.Therefore it is very important to improve its stability by the structural transformation.In our research,Arg in the entire sequence of Dybowskin-2CDYa was replaced by Lys according to the literature,and the transformed antimicrobial peptides was named RK6(SAVGKHGKKFGLKKHKKH).RK6 composed with 18 amino acid residues is a molecular weight of 2.015kD,the secondary structure is mainly of a-helix structure and the isoelectric point of 10.78,Instability index of 2.03 predictited by the HNN method,which showed that RK6 is more steady than Dy2 in the term of theory.RK6 gene got from SOE-PCR method was connected with yeast secretory expression vector pPICZa-A inorder to construct recombinant expression plasmid pPICZa-RK6 and then the plasmid was digestion with restriction endonuclease SacI and then linearized and transformed to Pichia pastoris X33 using method of electroporation.Positive transformants filtered by Zeocin were then identified by PCR method and then the right strains were induced by methanol.Supernatant of the fermentation liquid was tested by antibacterial effect measurement.The results showed that:the recombinant peptide RK6 had obvious antimicrobial action for both Gram-negative bacteria(E.coli E.coli),Gram-positive bacteria(Staphylococcus aureus S.au).We constructed a B.subtilis expression system further in order to investigate the application feasibility of RK6 in animal feed.Purpose one:recombinant proteins used in feed instead of antibiotic feed additives;Purpose two:Bacillus subtilis can be add in the feed as a probiotic;Objective III:fermentation culture(including Bacillus subtilis,RK6,and other proteins and enzymes)can be directly used in feed to reduce the production costs by ignoring the purification steps.In this study,we added the SacB promoter gene signal peptide sequence(SacR)of Bacillus subtilis and GST-Tag to upstream of RK6 inorder to get the fusion sequence GSR.Then the fusion sequence GSR was conected to Bacillus subtilis expression vector pHY300PLK so as to construct expression plasmids pHY-GSR,and then transformated to Bacillus subtilis WB600 and inducted by sucrose.The results showed that:peptide RK6 have been successfully expressed with antibacterial activity.We also add broth to chicks' fed,the animal experiments showed that:the fermentation liquid can be added in feed chicks for improved utilization of the feed,the growth ratio of meat chicks;chicks ?-IFN,SIg and IL-2 has significantly improved compared with controls(P<0.01).Conclusion of this study:1.Dybowskin-2CDYa and Dybowskin-2CDYb in the 11 peptides we got from the cDNA library of Rana dybowskii belong to a new family of antimicrobial peptide;2.Bacteriostatic experiment confirmed that:Dy2 showed obvious antimicrobial activity for Escherichia coli,Staphylococcus aureus glucose and other tested bacteria;3.We transformed Dybowskin-2CDYa into RK6 by amino acid substitution to eliminate its instability,transformation RK6 increased it's thermal stability while keeping antibacterial activity;4.RK6 had expressed in a Bacillus subtilis expression system.Fermentation broth can be used in animal feed to improve it's utilization ratio and FCR.Broth could also improve immune parameters of experimental animals significantly.