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

摘要

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 porcineβ-defensin114(PBD114)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 PBD114 in different breeds and tissues.For infection models,sixteen DLY pigs were divided into two groups and challenged either with sterile saline or E.coli K88.The recombinant protein PBD114(r PBD114)was obtained by using a heterologous expression system in E.coli.Results:PBD114 gene was highly expressed in tissues such as the intestine,liver,spleen,and thymus.Interestingly,the expression level of PBD114 gene was higher in the TP pigs than in the DLY pigs(P<0.05),and was significantly elevated upon E.coli K88 challenge(P<0.05).The nucleotide sequences of PBD114 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,PBD114 gene was successfully expressed in E.coli Origami B(DE3)and the molecular weight of the r PBD114 was estimated by SDS-PAGE to be 25 k Da.The r PBD114 was purified and mass spectrometry verified the protein as PBD114.Importantly,r PBD114 showed antimicrobial activities against E.coli DH5αand E.coli K88,and the minimal inhibitory concentrations(MICs)were 64 and 128μg/m L,respectively.Hemolytic and cytotoxicity assays showed that r PBD114 did not affect cell viability under physiological concentrations.Conclusions:PBD114 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 E.coli K88,suggested that it may serve as a candidate for the substitution of conventionally used antibiotics.