Expression,Purification And Activity Of Antimicrobial Peptide Mytichiytin-A In Chlamydomonas Reinhardtii

MytichitinA is an antimicrobial peptide derived from mussels. In 2014,it was discovered and purified by Sun Jingjing. The number of amino acid of MytichitinA was 55.The molecular weight of MytichitinA was 6.6 kDa.MytichitinA have three disulfide bonds.There was a certain inhibitory effect on gram positive bacteria and gram negative bacteria.In this study, three repeats of the MytichitinA gene were fused in tandem at first by genetic engineering techniques. The number of amino acids in 3×MytiA was 184. The relative molecular mass of 3×MytiA was 20.9 kDa.In this study, we We constructed a recombinant plasmid by 3 xMytichitinA and pBluescript SK+ vector and the recombinant plasmid was pBSK-3×MytichitinA. Then we constructed pBSK-Ble-FH-3×MytiA and Hsp/Rbcs-3×MytiA-Paro two expression vectors in Chlamydomonas reinhardtii. Both sets of vectors are suitable for transformation in Chlamydomonas reinhardtii. Ultimately,resistance screening and the western blotting results confirmed the presence of recombinant antimicrobial peptide 3×MytiA in five transgenic algal cells which were 1-7,1-23,1-90,1-99,1-130. The highest expression of the target protein was 1-90 transgenic algae by western blotting analysis. The target protein accounted for 0.31% of the Chlamydomonas reinhardtii total soluble proteins. The purification and enrichment of the target protein was achieved by Ni column affinity purification. The target protein obtained by vacuum freeze drying was used for analysis of the antibacterial activity. After vacuum freeze drying, the concentration of the recombinant antimicrobial peptide accounted for 13% of the total soluble protein. The tested strains were Escherichia coli 0157 (ATCC35150) , Staphylococcus aureus( ATCC 25923), Listeria bacteria Lester and Salmonella (ATCC 10467). The results showed that the antibacterial peptide 3×MytichitinA had antibacterial activity against Escherichia coli 0157 (ATCC 35150), Staphylococcus aureus (ATCC 25923),Listeria bacteria Lester and Salmonella(ATCC 10467). The minimal inhibitory concentration (MIC) assay showed that the recombinant protein 3 xMytichitinA had broad-spectrum antimicrobial activity. The MICs of Escherichia coli 0157 and Staphylococcus aureus were 60 ?g/mL, and the MICs of Listeria bacteria Lester and Salmonella were 80 ?g/mL. Finally, we resrarch the properties of the antibacterial peptide 3 xMytichitinA, it was found that its thermal stability is good and can withstand different temperatures and pH. For different proteases,such as pepsin, proteinase K and papain stability are very poor,but for trypsin ,it is very stability.In summary, the experimental results show that the antibacterial peptide MytichitinA series expression in Chlamydomonas reinhardtii by genetic engineering methods, and has antibacterial activity,which provide valuable experience for the acquisition of antimicrobial peptides.