Molecular Cloning And Recombinant Expression Of Macin From Mytilus Galloprovincialis

Antimicrobial peptides (AMPs) play a fundamental role in the innate immunity ofinvertebrates by exerting broad-spectrum microbicidal activity against pathogenic microbes. Theidentification and characterization of AMP genes is essential for the illustration of immune defensemechanisms and for disease control because of their potential use as therapeutic agents.In the present study, the cDNA encoding Macin was cloned from Mytilus galloprovincialis byexpressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. Thefull-length cDNA of Macin consisted of691nucleotides，containing a5`untranslated region(UTR)of276bp, a3`UTR of109bp with a poly(A) tail, encoding a polypeptide of101amino acids. TheN-terminus had the features consistent with a signal peptide as defined by SignalP analysis with aputative cleavage site located after position26. The deduced mature peptide was of75amino acidresidues with a theoretical mass of8.88kDa and a pI of8.38.Real-time PCR was employed to analyze the tissue distribution of Macin in Mytilusgalloprovincialis. The highest expression level was found in muscle and the lowest in gills. AfterVibrio anguillarum challenge, the Macin expression level in mantle of the mussels was increased6-fold compared to that of normal at24h, and restored to2.1-fold of normal level at48h. However,the expression level in muscle was down-regulated0.7-fold of normal level at24h, and thenup-regulated gradually with the4.5-fold compared with the normal level at48h.In order to determine the antibacterial activity of Macin in Mytilus galloprovincialis, themature peptide coding region was cloned into prokaryotic expression vector for expression. Theantibacterial experiment was processed after the purification and renaturation of the recombinantpeptide. Biological activity assay revealed that recombinant Macin exhibited significant activitytowards Micrococcus luteus, Bacillus subtilis, Enterobacter aerogenes, Vibrio parahaemolyticuswith the minimal inhibitory concentration (MIC) of1.1mg/mL,0.28mg/mL,1.1mg/mL and0.55mg/ml respectively.