Bioinformatics Analysis And Expression Research Of ?-defensin Gene From Chinese Giant Salamander

Beta defensin,with the stable structure,is a kind of small molecular peptide,which exhibit strong antibacterial and antiviral activities and perform a variety of biological activities.In the present research,based on the skin transcriptome database of Chinese giant salamanders Andrias davidianus,a novel ?-defensin gene was identified and characterised.We have used the real time quantitative PCR to analyse the mRNA expression pattern of ?-defensin during different tissues,developmental stages,bacterial and viral infections.Moreover,we also used the prokaryotic expression system to get the recombinant protein Ad-?-defensin and measured its bioactivities.The research results as follows:1.The full-length cDNA sequence of Ad-?-defensin was of 771 bp,consisting of 90 bp 5'-UTR,150 bp open reading frame and 531 bp 3'-UTR.The deduced protein was composed of 49 amino acids,with molecular mass of 5.52 kDa and a predicted pI of 9.23.The net charge of Ad-?-defensin is 5.87 at the pH 7 solutions.Based on the homology and phylogenetic analysis of Ad-?-defensin among other animals,it showed a close relationship with the amphibians.2.Real time quantitative PCR(qRT-PCR)analysis indicated that Ad-?-defensin showed the highest level in skin.It could be expressed during the larva stage.The expression of Ad-?-defensin could be up-regulated after bacterial and viral infections.The highest level of Ad-?-defensin could be observed at 24 hour after bacterial infection,while it was 120 hour after viral infection.3.The expression vector pET32a-?-defensin was constructed successfully.The recombinant protein was produced after IPTG induction in Escherichia coli BL21(DE3)and purified by Ni2+-chelating chromatography.From the bioactivities assay,the recombinant ?-defensin protein showed antimicrobial activities to both gram-negative and gram-positive bacteria.It showed a better inhibition activities to Vibrio alginolyticus,Escherichia coli,and Vibrio harveyi,and the minimal inhibitory concentration was about 19 ?g/mL.In conclusion,all these results indicated that the ?-defensin gene was a vital immune related gene in A.davidianus' sskin.It could play important functions during the response to bacterial and viral infections.Moreover,the recombinant ?-defensin protein could show a variety antimicrobial activities to different bacteria in vitro,which may be a potential effective bioactive peptides for exploiting the biological resources.