Cloning, Expression And Characterization Of Antimicrobial Peptides Of Anatolica Polita Borealis

The increasing of microbial resistance results in the intense demand of people on the research and development of new antimicrobial drugs.Antimicrobial peptides are a class of small molecule peptides, which can be obtained from mammals, insects, plants, marine organisms and so on. They are small peptides with anti-bacteria, anti-fungi, anti-cancer and some other biological activity. Here are four mechanisms of antimicrobial peptides: a. antimicrobial peptides interact with the cell membrane b. interaction between peptides and cell wall c. Interactions with nucleic acids d. peptides interact with cellular enzymes. The structure, physicochemical properties and other characteristics of antimicrobial peptides determine the action of antimicrobial peptides’ biological activity. Insects, the most diverse species of present biological sphere can survive harsh environmental conditions, which possess rich source of antimicrobial peptides with good stability, major water-soluble and broad range of antibacterial activity. Anatolica polita borealis inhabits in Junggar basin desert of Xinjiang, where seasonal temperature and temperature gap between day and night fluctuate violently and is extremely difficult for desert insects’ living. In recent years, with the deep study of antimicrobial peptides, antimicrobial peptides were found in addition to antibacterial, antifungal as well as the biological activity of tumor cells. In this study, experiments on Anatolica polita larvae with bacterial infections to produce an immune response have been done. The RNA from the larvae were extracted using Trizol and the antimicrobial genes were cloned by PCR. By using genetic engineering techniques of prokaryotic expression, affinity chromatography, Western blot method for antimicrobial peptides from A. polita were expressed, purified and characterized, and inhibition zone measuring and bacterial growth curve mode were used to detect their biological activity.1. In this study, antimicrobial peptide genes ApAMP55, ApAMP85, ApAMP1015 from A. polita have been cloned. ApAMP55 is 252 bp, which deduced encoding 83 amino acids residues including signal peptide with 18 amino acids, propeptide with 22 amino acids and mature peptide with 43 amino acids. ApAMP85 with 456 bp encodes 151 amino acids residues including signal peptide with 17 amino acids and mature peptide with 134 amino acids. ApAMP1015 is 387 bp encoding 128 amino acids including 19 amino acids signal peptide, propeptide with 35 amino acids and 74 amino acids mature peptide.2. By bioinformatics analysis on antimicrobial peptide genes ApAMP55, ApAMP85, ApAMP1015 from A. polita, We have got master composition, structure, hydrophobicity, charge and other physical and chemical properties of the three antimicrobial peptides, deducing their biological activity of three antimicrobial peptides in research.3. In this study, the prokaryotic expression system of antimicrobial peptides ApAMP85, ApAMP1015 and ApAMP55 have been successfully established, and the three peptides have been properly expressed.4. The expression conditions of antimicrobial peptide fusion protein TrxA-ApAMP1015 in E. coli BL21 was optimized with inducing at 0.1mmol/L IPTG for 6h at 25℃and 150 rpm, which lay the foundation for the application of insect antimicrobial peptides.5. A preliminary exploration and research on biological activity of the antimicrobial peptides showed that the recombinant antimicrobial peptide TrxA-ApAMP1015 can inhibit the growth of E. coli, ApAMP55 and ApAMP 85 have not showed any effective antimicrobial activity, which antimicrobial activity need to be further explored. These studies lay the foundation on the application and further study of antimicrobial peptides from A. polita.