| The existence and extended diversity of antimicrobial peptides has been revealed in mussel in recent years. They present in the body fluids of the mussel at a great abundance and were characterized as the small cysteine-rich cationic peptides. According to the common features of their primary structure, these molecules are classified into four groups, e.g., defensins, mytilins, myticins and mytimycin.Myticin A is one of the member of Myticins which consisting of. 40 amino acid residues. It showed an effect of inhibition to the growth of gram-positive strains, but has little or no effect to gram-negative strains and fugi. For lower natural production and higher cost of synthesis, the genetic engineering and recombinant expression would become the appropriate way to study and exploit the peptide.In the present study, cDNA of Myticin A was amplified by RT-PCR from the total RNA of mussel body fluid. We constructed the recombinant plasmid of pPIC9K-Myticin A. Myticin A was expressed successfully in the methylotrophic yeast pichia pastoris. The purification was involved in two steps including ion-exchange column chromatography on CM-Sepharose and Sephadex G25. The recombinant peptide was tested in vitro bioassay and was shown a remarkable inhibition against the gram-position bacterial strains. In addition, it inhibited the growth of several tumor cell lines.This research describes: (1) The cloning of cDNA of Myticin A and construction of the sequencing vector; (2) Construction of recombinant vector pMDIS-Myticin A and expression vector pPIC9K-Myticin A; (3) Expression of Mussel Myticin A in the methylotrophic yeast Pachia pastoris; (4) Separation and purification of recombinant Myticin A with column chromatography and assessment of its biological activities.Methods:1. Total RNA was isolated from adult mussel body fluid, using mRNA to construct acDNA library. Reverse transcriptional PCR was used to obtained Myticin A gene fragment.2. A recombinant plasmid of pMDIS-Myticin A was constructed and sequenced, it was transformed into E.coli TOP 103. The Myticin A gene was inserted into the P. pastor is expression vector pPIC9 and pPIC9K respectively. The pPIC9K-Myticin A was transformed into electrocompetent cells of P. pastoris strain GS115. The transformants were selected out and the recombinant peptide was expressed. The expressed sample was analyzed by Tricine-SDS-PAGE.4. The expressed sample was purified by CM-sepharose and Sephadex G25 column chromatography.5. The biological activities were assessed by the inhibition of the growth of bacteria and turner cell lines in vitro.Results:1. The purity of RNA was estimated by the absorbance ratio at Aa6o/A2go run, and was confirmed by agarose gel electrophoresis. The cDNA was obtained by RT-PCR and yielded the gene fragment Myticin A. DNA sequencing was carried out and proved to be identical to the natural Myticin A.2. The recombinant vector pMDIS-Myticin A was obtained by the ligation of Myticin A gene fragment to pMD18-T. The correct insertion was confirmed also by DNA sequencing.3. The Myticin A was inserted into the P. pastoris expression vector pPIC9K, and was verified by BamH I and EcoR I digestion and DNA sequencing as well, Electrocompetent cells of P. pastoris strain GS115 were transformed with the Sal I linearized plasmid pPIC9K-Myticin A. The stable recombinant P. pastoris strains were selected by G418 resistance. Myticin A was successfully expressed as identified by Tricine-SDS-PAGE.4. The recombinant Myticin A was firstly purified by CM-sepharose column chromatography and the active fraction was collected, and was further purified bySephadex G25. Purified sample was analyzed by Tricine-SDS-PAGE. 5. Myticin A shows a strong inhibitory effect against the growth of gram-positive bacteria, specially to the Bacillus megaterium. It also inhibited the growth of turner cell lines including SW1990,L929 and LoVo.Conclusion:An antimicrobial peptide, Myticin A , was cloned from the body fluid of mussel and...
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