Molecular Cloning, Expression Of Big Defensin And G-type Lysozyme Genes From Scallops, And Antimicrobial Activity Of The Recombinant Proteins

Scallop aquaculture is a big industry and contributes enormously to the economicdevelopment of coastal provinces in China. Since the summer of 1997, large-scalemortality of cultured scallop has caused catastrophic losses to scallop aquaculture,which resulted in the production decreasing drastically. The durative outbreak ofdiseases has stimulated intensive efforts for the development of better healthmanagement strategies and characterization of original immune efforts for diseasecontrol.The identification and characterization of genes involved in scallop immuneresponses are now considered to be essential for the elucidation of immune defensemechanisms and disease control because of their potential use as therapeutic agentsand genetic improvement biomarkers on disease-resistant strain selection.Antimicrobial effectors constitute the first line of innate immunity for scallop exposedto various potential pathogens in the aquatic environment by exerting broad-spectrummicrobicidal activity.The first mollusk big defensin (designated AiBD) cDNA was cloned from bayscallop Argopecten irradians by expressed sequence tag (EST) and rapidamplification of cDNA ends (RACE) techniques. The full-length cDNA of AiBDconsisted of 531 nucleotides with a canonical polyadenylation signal sequenceAATAAA and a poly(A) tail, encoding a polypeptide of 122 amino acids. Consistentwith other known big defensin sequences, AiBD was firstly synthesized as aprepropeptide. After cleavage of the signal peptide, the proregion was processed byKex-2-like proteases and resulted in the mature peptide with a theoretical mass of9.22 kDa and a pI of 9.81. The expression of AiBD in various tissues was measured byusing Northern blotting analysis. mRNA transcripts of AiBD could be detected inhaemocytes of unchallenged scallops. The temporal expression of AiBD inhaemolymph after Vibrio anguilarum challenge was recorded by quantitative real timePCR. The relative expression level of AiBD in haemolymph was up-regulated evenlyin the first 8 h, followed by a drastic increase, and increased 131.1 fold at 32 h postinjection. These results indicated that AiBD could be induced by bacterial challenge,and it should participate in the immune responses of A. irradians. Biological activityassay revealed that recombinant AiBD could inhibit the growth of both Gram-positiveand Gram-negative bacteria, and also showed strong fungicidal activity towards theexpression host.The genomic structure of the first invertebrate G-type lysozyme from Chlamysfarreri (designated CFGLys) was obtained by genome walking approach. TheCFGLys gene was 8131 bp in length, coded for 829 bp and 200 deduced amino acidresidues. By comparison to the cDNA sequence and its translation product, the codingregion was found separated in six exons of 55 bp,60 bp,90 bp,113 bp,145 bp and140 bp, respectively. The introns range in size from 592 to 2161 bp, and havetraditional spliceosomal intron 5′-GT donor and 3′-AG acceptor sites for splicing.Analysis of the 5′-flanking sequence of CFGLys gene by TRANSFAC softwarerevealed several putative binding sequences for transcriptional factor found in hostdefense genes of other animals, including sequences similar to the NF-κB, OCT-1,AP-1 and NF-IL6. The expression of CFLysG mRNA in various tissues was measuredusing Northern blot analysis. mRNA transcript of CFLysG was most abundantlyexpressed in the tissues of gills, hepatopancreas and gonad, to a lesser degree in thetissues of haemocytes and mantle, while undetectable in the adductor muscle. Theseresults suggested that CFLysG could possess combined features of both the immuneand digestive adaptive lysozymes. In order to determine the in vitro lytic activities ofCFLysG, the mature peptide coding region was cloned into Pichia pastoris forheterogeneous expression. Recombinant CFLysG showed inhibitive effect on thegrowth of both Gram-positive and Gram-negative bacteria with more potent activityagainst Gram-positive bacteria, which demonstrated the involvement of CFLysG inthe innate immunity of C. farreri.Recombinant expression of AiBD and CFLysG made it possible to furthercharacterize their biological activities, three dimensional structures and immunefunctions, and also provided potential therapeutic agents for disease control inaquaculture.