Cloning, Expression And Antimicrobial Activity Analysis Of A Novel Anionic Antimicrobial Peptide SCY2 From Scylla Serrata

A novel antimierobial peptide SCY2 cDNA was cloned from the seminal plasma of the mud crab,Scylla serrata(Forsk(?)l,1775) by using a degenerated reverse transcriptase(RT)-PCR and rapid amplification of cDNA ends(RACE).The full-length cDNA of SCY2 consisted of 947 nucleotides,encoding a polypeptide of 100 amino acids.The mean molecular weight of mature peptide is 10925.4 Da.The identity of amino acid sequences between the homologous cDNA and seygonadin cDNA was 65.08%and the theoretical pI of the mature peptide is 4.84,which suggests that it is also an anionic molecule as scygonadin(pI 6.09).The amino acid sequence of it is different from any other reported antimicrobial peptide,indicating that it is a new gene.Genomic DNA sequence of SCY2 was determined by PCR and inverse PCR,consisting of 2607 bp including two introns and three exons similar to the genomic organization of scygonadin.A TATA box and a cap was found at the location where 5' site started for transcription.In addition,several consensus-binding motifs for transcription factors were also found.Using the semi-quantitative RT-PCR and northern blot methods,SCY2 transcripts were investigated in various tissues including exoskeleton,subcuticular epithelia,gills, heart,hepatopancreas,stomach,muscle,eyes,female and male reproductive apparatus. The result showed the SCY2 gene was only expressed in the male reproductive apparatus.While testing on mRNA transcripts of either SCY2 or scygonadin,respectively in bacterially challenged mud crab,it was found that there was no significant change of SCY2 and scygonadin mRNA abundance in all of the tested tissues,suggesting that bacterial infection can not induce the transcription of both SCY2 and scygonadin genes.The antibacterial activity of SCY2 was evaluated on ten strains of bacteria using the recombinant fusion SCY2 expressed in Escherichia coli system.The SCY2 recombinant pTrc-CKS vector was constructed,the fusion protein was expressed in Escherichia coli with IPTG induction.The expressed protein existed in the inclusion body which was denaturalized,dissolved and recovered.The fusion proteins were purified by Ni-NTA column,The recombinant SCY2 after cleavage with 3C protease exhibited obvious antibacterial activities against several bacteria tested including Aeromonas hydrophia,Staphylococcus aureus,Corynebacterium glutamicum.Thus, SCY2 is a male-specific anionic antimicrobial pepetide transcribed by a new gene and belongs to a family of scygonadin but might play a different role in innate immunity in Scylla serrata.