Construction of recombinant chicken AvBD5-AvBD10 double molecule protein and analysis of its physical and chemistry characterizations.

Chicken antimicrobial peptides,belong to β-defensins,are essential components of innate immunity and play significant roles in innate immunity in chickens.The cDNA of chicken avian β-defensins 10(AvBD10) gene was sub-cloned into Sal Ⅰ and Not Ⅰ sites of AvBD5-pGEX-6p-1 vector to construct recombinant plasmid AvBD5-pGEX-AvBD10.The recombinant plasmid was transferred into Escherichia coli BL21.Then the bacteria was induced with IPTG at 37 ℃ and different time,and exogenous expression was detected by SDS-PAGE.Results showed that a 36 kD protein which was equal to chicken AvBD5-AvBD10 protein in molecular weight was expressed in E.coli BL21.The production of AvBD5-AvBD10 accounted for approximately 35% of the total protein.The recombinant fusion protein was purified and expressed as insoluble body.Two bacteria,E.coli and pathogenic Streptococcus of mid-log(108 cfu/mL) were used to evaluate antibacterial activity of the recombinant fusion protein by inhibition zone assaying.The results showed that the recombinant fusion protein exhibited anti-E.coli.and anti-pathogenic Streptococcus activity,and had high stability at various temperatures-70℃～100 ℃ and pH 3～12.