Constitutive Expression Of AvBD2 In Pichia Pastoris And Optimization Of Its Fermentation Conditions

Avain beta-defensin 2 mature peptide?AvBD2?,a small cysteine-rich cationic peptide,is composed of 39 amino acids and plays an important role in the innate immunity and acquired immunity of chicken.It is widely expressed in the digestive tract,and has a strong antimicrobial activity.Moreover,it has a broad spectrum of antimicrobial activity without residues and bacteria resistance.AvBD2 had been expected to become a substitute of the traditional antibiotics which could be used in livestock and poultry industry.In this study,AvBD2 and the glyceraldehyde-3-phosphate dehydrogenase constitutive expression vector?pGHK??was employed to constructe Pichia pastoris constitutive expression system of AvBD2.Then,the activity of expression was analyzed and the fermentation conditions were optimized by response surface method.These data would provide foundation for the fermentation and industrial application of AvBD2.1?Constitutive recombinant and expression of chicken AvBD2 in Pichia pastorisAccording to AvBD2 gene sequence in NCBI?access number: NM₂04992?and Pichia pastoris' s codon bias,pUC57-AvBD2 cloning vector was synthesized.After the double digestion of pUC57-AvBD2 and pGHK ? with EcoR ? and Not ?respectively,target fragments were used to construct recombinant plasmid pGHK?-AvBD2.The recombinant plasmid was linearized by Sac?and Bgl ? to remove Amp resistance gene,and was electrotransformated into Pichia pastoris?GS115?,then high-level expression strains were screened by G418-resistant and concentrations of expression protein.Tricine-SDS-PAGE and Western Blot results showed that the proportion of target protein,a relative molecular mass about 5.8kDa.2?Antimicrobial activity of recombinant AvBD2The high expression strain was inoculated into a fermentation medium and cultured overnight,then antibacterial and hemolytic activity of concentrated supernatants were analyzed.The concentrated expression supernatant was used for antimicrobial assays by agar diffusion method,the results showed that the the recombinant protein had significant antibacterial activity against E.coli,Enterococcus faecalis,Enterobacter aerogene,Pseudomonas aeruginosa,Staphylococcus aureus and Pasteurella.The protein of AvBD2 was diluted to 100,250 and 500 mg/L of the three gradient with sterile PBS and fresh chicken erythrocytes diluted to 2%-3%,corresponding respectively hemolysis rate was 0.74%,1.12% and 2.17 %.3.Fermentation conditions optimization of recombinant AvBD2Growth curve of high-level expression strain was measured in the YPD broth.The result showed the strain viability was the most preferable after the seed had been cultured by 22 h.Firstly,three factors such as fermentation temperature,inoculum dose and liquid volume were optimized separately,and the appropriate levels were selected.Secondly,the these conditions were optimized by response surface medthod,the results shown the concentration of fermentation protein was increased by 20%when GS115-AvBD2 was induced in YPD at 27.8 ?,with addition of 14.44%inoculum and liquid volume 8.43%.In summary,the recombinant Pichia pastoris contained AvBD2 gene was built by the constitutive expression vector pGHK?,Tricine-SDS-PAGE and Western Blot showed that the target protein had been expressed in the secretory supernatant with a relative molecular mass about 5.8kDa.Antimicrobial activity and hemolytic studies show that AvBD2 had significant antibacterial activity against pathogens with a low hemolytic activity.After optimizing the fermentation conditions,the concentration of fermentation protein was increased by 20% when GS115-AvBD2 was induced in YPD at 27.8 ?,with addition of 14.44% inoculums and liquid volume 8.43%.These data provide a theoretical foundation for industrial scale-up production of chicken AvBD2 which has the potentiality used asa new food additive against bacteria in poultry farming.