Molecular Characterization And Recombinant Expression Of Novel Antimicrobial Peptides From Mytilus Coruscus

Higher animals and insects are always preferred when it comes to the study ofantimicrobial peptides, while recently researchers shift their focus to marine animals. Weobtained two kinds of antibacterial constituents by isolating the hemolymph sample ofMytilus coruscus, one of the most commercially important bivalves in Zhejiang province.They were named Myticusin-1and Mytichitin respectively. Partial N-terminal sequencesdetermined by Edman degradation were searched against the dbEST database. The matchedEST sequences were used as the templates to design specific primers for PCR amplification.Based on the characteristics of reverse transcription and the reverse transcriptase, a solutionnamed “two-step” method was introduced into the research, which was used to obtain thecDNA3’ end untranslated region. We obtained the5’ end untranslated region ofMyticusin-1cDNA from M. coruscus hemocyte cDNA library constructed by thepredecessor of the lab, but failed in the other one. The cDNA of Myticusin-1has beenconfirmed by sequencing. Its ORF encodes a precursor containing a signal peptide of24residues and a mature peptide of104residues. Mytichitin mature peptide consists of56aa,including6cysteines.Real-time qPCR was employed to establish the expression patterns of Myticusin-1andMytichitin transcripts in tissues. The results suggested that the hemocyte serves as the maintissue for expressing Myticusin-1transcripts and the expression level reached the peak at36h after microbial challenge. As for Mytichitin, gonad appears as the main tissue and itreached the highest expression level at12h post-injection.We transferred the gene encoding Myticusin-1and Mytichitin mature peptides intopET-28a(+) expression vector successfully. Recombinant expression of Mytichitinsucceeded. Further research is needed to screen for optimized fermentation condition ofrecombinant expression of Myticusin-1.The research above laid the foundation for further development of new molecule drugof antimicrobial peptides and finally realizing industrial production.