Cloning, Expression And Antibacterial Activity Analysis Of Beta Defensin From Intestinal Of Grass Carp

Defensin is not only as a defense barrier which plays an important role against microbial invasion, but also an important component part of innate immunity system. Defensin is a small peptide with the molecular weight of3.5～6kDa. which has a broad spectrum of antibacterial activity. Defense is the basis of natural host recognition of microbes which usually consists of29～54bp amino acid, with the characters of cysteine and arginine rich, strong cationic and thermal stability. Defense not only has various cytotoxicity and bioactivity to bacteria, fungus, virus, parasite, but also has Immune cell chemotaxis which can regulate the immune system of organisms.High conserved sequence was found among beta-defensin-like2（defbl2） mRNA of Danio rerio, beta-defensin-like2（defb12） mRNA of Oncorhynchus mykiss. beta-defensin gene of Garrupa （GenBank association number:NM₀01081554.1. NM₀01195168.1. JN698964.1respectively）. And specific primers were designed base on this conserved resign. Then total RNA were obtained as template.And about160bp amplicons of beta-defensin-like gene was obtained by RT-PCR. The amplicon was cloned into pET-32a vector. And high-efficiency prokaryotic expression vector named pET32-GBD was structured by double digestion. SDS-PAGE and Western Blot showed that about22kDa recombination peptide with antigenicity was obtained by prokaryotic expression. About95%recombination protein was purified by Ni2+affinity chromatography. After enterokinase digestion, we analyzed the antimicrobial spectrum of5KD recombination small peptide. The results demonstrated that it could restrain the growth of Salmonella, Aeromonas hydrophila. Pasteurella, Escherichia coli. Streptococcus, Staphylococcus aurens, Myxococcus pisciocola. The results proved that intestinal tract original beta-defensin of grass carp possessed broad-spectrum antimicrobial activity to Gram-positive and Gram-negative bacteria.