Molecular Cloning,Expression And Antibacterial Activity Of Recombinant Proteins For β-defensin Gene In Yellow Catfish(Pelteobagrus Fulvidraco)

Beta-defensins are a large family of vertebrate-sepecific antimicrobial peptides(AMPs)and are widespread in the epithelial cells,neutrophils and macrophages and play an important role in innate immune system against those invading pathogens.Yellow catfish(Pelteobagrus fulvidraco)is an important commercial freshwater fish in China.It is favoured and welcomed by many consumers due to its delicious meat quality and excellent taste.In recent years,kinds of bacterial diseases,such as red-head disease,ascites disease,ulcerative syndrome,have been discovered in artificial aquaculture of yellow catfish and have led to serious economic losses.In order to understand the function of β-defensin gene(Pf＿ΒD)in innate immune system of P.fulvidraco,the part of Pf＿ΒD gene c DNA sequence has been successfully cloned by molecular biology technology in this study.At the same time,we analyzed the expression profiles changes of Pf＿ΒD gene m RNA in various adult tissues and during the embryonic and early developmental stages,and detected the changes of m RNA expression levels of Pf＿ΒD gene in five tissues after injection of killed Aeromonas hydrophila.Finally,we identified the antibacterial activity of recombinant proteins of Pf＿ΒD gene.The main results obtained were as follows:1.Molecular cloning and sequence analysis of Pf＿ΒD gene c DNAThe partial c DNA sequence of Pf＿ΒD gene was 708 bp(Gen Bank accession No.KY091312),and contains 225 bp open reading frame(ORF)and a 483 bp 3′ untranslated region(UTR).The 225 bp ORF of the Pf＿ΒD gene encoded 74 amino acids,including a signal peptide of 25 amino acids and a mature peptide of 49 amino acids.The deduced amino acid sequence of Pf＿ΒD gene based on ORF was highly similar to the β-defensin-4gene of rainbow trout(68%).2.The expression analysis of Pf＿ΒD geneThe results of real-time quantitative PCR showed that the expression of Pf＿ΒD m RNA had the highest level in swim bladder,followed by a moderate level in heart,gill,liver and skin.During the embryonic and early development stages,the expression of Pf＿ΒD m RNA had the highest level at multicellular stage and kept at a high level until somite appearance stage,after which kept at a moderate level until 2 days post-hatching(dph).The extremely low level of expression appeared at 3 dph,and kept the steady state until 30 dph.After injection with killed Aeromonas hydrophila,compared with the control group,the expression levels of Pf＿ΒD m RNA in all of five tissues had a significant up-regulation.There was a significant up-regulated expression at 12 h after injection and peaked at 24 h after injection in swim-bladder.In heart,the peak of expression appeared at 12 h after injection and kept at a more stable level all the other times.The significant up-regulated expression appeared at 3 h after injection and the peak appeared at 48 h after injection in gill.In liver,the expressions were kept at a stable level except for an obvious down-regulation at 48 h after injection.In the tissue of skin,the expression was significantly up-regulated to the peak at 6 h after injection,and was maintained at arelatively during the remaining time.3.The expression of Pf＿ΒD gene in Escherichia coil and the identification of antibacterial activity of recombinant productsThe recombinant prokaryotic expression vector PET-32a-Pf＿ΒD was constructed successfully.A large number of recombinant proteins were expressed at 37℃,6 h and 1.2m M IPTG and were detected as soluble proteins.In the eluent with imidazole,the recombinant proteins about 4500 μg/m L were purified with a high purity.The antibacterial activity of the products was tested by oxford cup method,the results showed that recombinant proteins had obvious inhibitory effect on Staphylococcus aureus,E.Coli DH5α,Aeromonas hydrophila,Edwardsiella ictaluri and Flavobacterium columnari.The results proved that β-defensin of yellow catfish had a broad-spectrum antibacterial function to Gram-positive and Gram-negative bacteria.