High-level Constitutive Expression And Production Optimization Of Chicken AvBD9in Pichia Pastoris

Avain beta-defensin9mature peptide (AvBD9) was a small cysteine-rich cationic peptide composed of41amino acids, played an important role in the innate immunity and acquired immunity of chicken. It was widely expressed in the digestive tract, and had strong antimicrobial activity. Moreover, its broad spectrum of antimicrobial activity, no residue and no bacteria resistance made by and so on, AvBD9was expected to become a substitute of the traditional antibiotics and use in livestock and poultry industry.In this study, AvBD9was chosen as the research object, and the glyceraldehyde-3-phosphate dehydrogenase promoter pGAP was employed to direct the constitutive expression of recombinant AvBD9in Pichia pastoris. First, the constitutive expression vector was constructed with the GAP promoter by molecular biology techniques, and evaluated whether the vector was available. The P. pastoris constitutive expression system of chicken AvBD9was constructed, then the fermentation conditions and its antimicrobial activity were investigated, specific contents and research results were showed as follows:1. Construction and evaluation of Pichia pastoris constitutive expression vectorBased on the gene sequence of glyceraldehyde-3-phosphate dehydrogenase promoter in GenBank (accession number:U62648), a pair of primers GAP1(Sac I restriction sites in5’end) and GAP2(BamH I restriction sites in5’end) was designed, pGAP was amplified by PCR and connected to the pMD19-T Simple Vector. After the double digestion of pMD19-T Simple Vector-pGAP and pPIC9K by Sac I and BamH I, target fragments were ligated to construct the constitutive expression vector pGHK a Staphylococcus nuclease gene nucA was cloned to pMD19-T Simple Vector, then reporter gene nucA obtained after the double digestion by EcoR I and Not I, was inserted into pGHK a which was digested in the same way in order to construct the recombinant plasmid pGHKa-nucA. By linearization, the plasmid was electrotrans-formated into P. pastoris GS115. Tricine-SDS-PAGE analysis showed that the reporter protein could be secreted by the recombinant yeast, and contained42.08%of the total protein in the supernatant.2. Constitutive secretory expression of chicken AvBD9in Pichia pastorisAccording to AvBD9gene sequence in GenBank (accession number:AY534894.1) and P. pastoris codon bias, AvBD9mature peptide gene sequence which in turn contained EcoR I restriction sites, coding sequences of Kex2cleavage sites AAAAGA, TAA sites and Not I restriction sites from5’end to3’end was designed and synthesized. After the double digestion of pUC57-AvBD9and pGHK a with EcoR I and Not I respectively, target fragments were used to construct recombinant plasmid pGHK α-AvBD9. The plasmid was linearized by Sac I and Bgl II to remove Amp resistance gene, and was electrotransformated into P. pastoris GS115, then high-level expression strains were screened by G418-resistant and concentrations of protein expressed from different strains. Tricine-SDS-PAGE showed that target protein in the secretory supernatant, with a relative molecular mass of about6kDa, contained11.38%of total supernatant proteins. The recombinant AvBD9was secreted into the culture medium at about66.18mg/L.3. Production optimization of of recombinant AvBD9and its antimicrobial activityHigh-level expression strain obtained above, was measured growth curve in the YPD broth. The result showed, when22h cultures were used as the seed, the strain viability was preferable. First, three factors such as fermentation time, inoculum dose and liquid volume were analyzed separately, and the appropriate levels were selected. The result of orthogonal tests showed, in the conditions of liquid volume of10%and inoculum dose of10%, after recombinant yeast Y3was cultured for48h, the expression level of protein came to the highest. The concentrated expression supernatant was used for antimicrobial assays. The results showed that the expression supernatant was active against pathogenic microbes contained Escherichia coli, Salmonella paratyphi A, Salmonella pullorum, Pseudomonas aeruginosa, Enterococcus faecalis, Enterobacter cloacae and so on.In summary, a novel constitutive expression vector pGHKa used for P. pastoris was successfully constructed in this study. The recombinant yeasts contained AvBD9gene were successfully obtained by the plasmid pGHKa, recombinant AvBD9was constitutively secreted into the supernatant at about66.18mg/L. The expression conditions in shake flask were optimized, and when recombinant yeast Y3was cultured for48h in the conditions of liquid volume of10%and inoculum dose of10%, protein concentration in the fermentation broth came to the highest. The antimicrobial assays showed AvBD9supernatant could have antimicrobial activity against several pathogenic microbes. These data can be used as the foundation for the further study on the chicken AvBD9, its fermentation production and industrial application.