Differential expression, molecular cloning, and characterization of porcine beta defensin 114.

BACKGROUND

β-defensins have attracted considerable research interest because of their roles in protecting hosts from various pathogens. This study was conducted to investigate the expression profiles of the porcine β-defensin 114 () in different breeds and in response to infections. Moreover, the function of PBD114 protein was partially investigated.

METHODS

Six Tibetan pigs (TP) and six DLY (Duroc×Landrace×Yorkshire) pigs were slaughtered to explore the expression profiles of in different breeds and tissues. For infection models, sixteen DLY pigs were divided into two groups and challenged either with sterile saline or K88. The recombinant protein PBD114 (rPBD114) was obtained by using a heterologous expression system in .

RESULTS

gene was highly expressed in tissues such as the intestine, liver, spleen, and thymus. Interestingly, the expression level of gene was higher in the TP pigs than in the DLY pigs ( < 0.05), and was significantly elevated upon K88 challenge ( < 0.05). The nucleotide sequences of from Tibetan and DLY pigs was identical, and both showed a 210-bp open reading frame encoding a 69-amino acid mature peptide. To explaore the function of PBD114 protein, gene was successfully expressed in Origami B (DE3) and the molecular weight of the rPBD114 was estimated by SDS-PAGE to be 25 kDa. The rPBD114 was purified and mass spectrometry verified the protein as PBD114. Importantly, rPBD114 showed antimicrobial activities against DH5α and K88, and the minimal inhibitory concentrations (MICs) were 64 and 128 μg/mL, respectively. Hemolytic and cytotoxicity assays showed that rPBD114 did not affect cell viability under physiological concentrations.

CONCLUSIONS

is an infection response gene that is differentially-expressed between different porcine breeds and tissues. The antimicrobial activity of PBD114 protein, against pathogens such as the K88, suggested that it may serve as a candidate for the substitution of conventionally used antibiotics.