Eukaryotic Expression Of Porcine Antimicrobial Peptides And Their Antimicrobial Activity In Vitro

In our study, porcine antimicrobial peptides (AMPs) were found by using digital gene expression profile, and detected through real-time PCR. The aquired AMPs were expressed in Pichia pastoris, then purified through ProteinlsoTM Ni-NTA resin, Strong ion exchange resin Poros(?) 50 HS and dialysis, the purity of recombinant protein reached more than 95%. Antimicrobial activity assay, MTT assay, transmission electron microscopy and real-time PCR technology were applied to detected the antibacterial activity, cell toxity and anti-inflammatory effect. The results showed that:1.15 AMPs were abtained from the infected PRRSV pig by bioinformatics, DGEP databases and real-time PCR.2. The codon-optimized porcine AMPS DNA carrying restriction sites was synthesized, the recombinant plasmid were constructed, linearized by SacI, transformed into the Pichia pastoris strain and expressed using shaking flasks. The recombinant yeast which expressed CST11、NK-lysin and β-defensin-2 were obtained. The ProteinlsoTM Ni-NTA resin was used to capture the soluble porcine AMPS through its His-tags, a strong ion exchange resin Poros(?) 50 HS was chosen for the second purification, the final purification yield of CST11、 NK-lysin and β-defensin-2 was 30 mg/L,12 mg/L and 9 mg/L from the original harvested supernatant respectively.3. Antimicrobial activity analysis showed:CST11 showed antimicrobial activity against E. coli, S. aureus, S. epidermidis and B. subtilis, and suppress the activity of papain, E. coli and S. aureus treated with CST11 showed Shorter or smaller of cells, shrunk and discontinuous of membrane, outflow of cell content; NK-lysin showed antimicrobial activity against E. coli, S. aureus, S. epidermidis, B. subtilis, B. pumilus and M. luteus; β-defensin-2 showed antimicrobial activity against E. coli, S. aureus, S. epidermidis, B. subtilis and B. pumilus.4. The ultrastructure of SMMC-7721 cells treated with NK-lysis exhibited exhibited the entire shrinkage, local narrowing of lamellipodia with microvilli and marked alterations in cell-cell contacts among neighboring cells; the ultrastructure of SMMC-7721 cells treated with β-defensin-2 exhibited the boundaries between cells vague, and lamellipodia and filopodia on cell membrane decrease. NK-lysin and P-defensin-2 can inhibit the expression of fascin 1 in SMMC-7721 cells5. NK-lysin and β-defensin-2 downregulate the expression of IL-1β, IL-6, TNF-α, iNOS and COX-2 in LPS induced murine macrophage cell line RAW264.7 cells, the results showed NK-lysin and β-defensin-2 possessed anti-inflammatory activity.