Isolation, Characterization And Gene Cloning Of Mytilin From Mytilus Coruscus

Mytilius coruscus mainly spreads all through the East China Sea and is an important aquaculture specie in Zhejiang Province. Based on the immune factor-antimicrobial peptide in their body, M. coruscus has been an interesting subject to researchers. To understand the molecular composition and characteristics of mytilin of M. coruscus, three peptides with antibacterial activity were purified from M. coruscus serum by high performance liquid chromatography (HPLC) combinning with gel filtration. The three peptides exhibited complementary antimicrobial properties against both gram-positive and gram-negative bacteria. The mass and the N-terminal sequences of these three peptides were analyzed by a combination of Edman degradation and Mass Spectrometry. Based on the results of sequential BLAST, these antibacterial peptides from M. coruscus serum belong to mytilin family and are named mytilin-1, mytilin-2 and mytilin-3, respectively; The molecular mass of these antibacterial peptides are 3885.17 Da,3993.26 Da and 3991.39 Da, respectively. Among them, mytilin-1 was characterized as a 34-residue peptide including eight cyctines formed four disulfides. The cDNA sequence coding for the mytilin-1 precursor was obtained by screening PCR from M. coruscus hemocyte cDNA library. The precursor of mytilin-1 contains a putative signal peptide of 22 residues, a processing peptide sequence of 34 amino acids, and a C-terminal extension of 46 residues rich in acidic residues.To obtain the expressed sequence tags (ESTs) and full-length sequences of potential functional-genes, the total RNA of M. coruscus hemocyte was extracted with Trizol reagent, and purified in chromatography with oligo(dT) cellulose. In gateway technology, a high-quality cDNA library of M. coruscus was constructed with pCMV sport6 as the vector. A number of clones were randomly-selected, sequenced, and online-analyzed with BLAST. The result showed that among the 28 random clones, four were characterized as rRNA genes, seven were unknown genes (presumably novel ones), and 17 were functional genes (coded for proteins including dehydrogenases, cytochrome oxidases, heat shock proteins, metal-binding proteins and actins etc.), some of them were immunity-related. Followed by large-scale sequencing of the library, two kinds of antimicrobial peptide mytilin gene sequence named mytilin-A and mytilin-B was received, the remaining sequences were submitted to NCBI dbEST database.In addition, to investigate the molecular diversity and the sequence characterization of mytilin and myticin from M. coruscus, the cDNAs of mytilin and myticin were cloned respectively by PCR method using M. coruscus hemocyte cDNA library as template. As a result, eight deferent mytilin cDNAs and nine deferent myticin cDNAs were cloned from M. coruscus hemocyte cDNA library, respectively. The precursor translated from mytilin cDNA contains a putative signal peptide of 22 residues, a processing peptide sequence of 34 amino acids, and a C-terminal extension of 46 residues rich in acidic residues; while the precursor translated from myticin cDNA contains a putative signal peptide of 20 residues, a processing peptide sequence of 40 amino acids, and a C-terminal extension of 37 residues. Both mytilin and myticin of M. coruscus are characterized by abundant cationic amino acids and eight cysteins which formed four disulfides. Sequence analysis of the coding frame showed that the variation loci of these two kinds of antimicrobial peptide mainly occurred on mature peptide region, which might result from direct interaction of these peptides with the surrounding pathogens in the changing environment and thus become a likely cause of molecular adaptation of certain amino acid sites in mytilin and myticin variants.In summary, by using molecular biology, biochemistry, mass spectra etc, we have investigated antimicrobial peptide composition and peptide sequence of the cDNA, these studies lay the foundation for screening M. coruscus immune-related genes and to explore the mechanism of molecular diversity of immune-related cytokines.