Discovery And Expression Of Novel Antimicrobial Peptides Based On Metagenomic Library And The South China Sea Sediment

Antimicrobial peptide is a class of biological peptide with functions of antiviral,antifungal.and antibacterial activities.It is an important potential substitute of conventional antibiotic in a wide range of applications in biomedicine,food preservation,and animal husbandry green farming.In this paper,a macrogenomic library was constructed based on the marine sediments of the South China Sea.A new type of antimicrobial peptide with industrial application value was developed by using the screening method based on antibacterial peptide function.The primary structure was analyzed by amino acid sequencing technology.Subsequently,the extracellular expression system of the novel antimicrobial peptide was optimized by genetic engineering technology and Bacillus subtilis expression system.Results presented in this thesis can be summarized as follows:-Metagenomic library DNA was extracted,purified and obtained from marine sediments in the South China Sea using metagenomic technology.-Based on the DNA of the marine sediment macrogenomic library of the South China Sea,a metagenomic library based on the B.subtilis expression system was constructed.-The positive clones producing the inhibition zone from the marine sediment macrogenomic library of the South China Sea were selected from the antibacterial peptide functional plate,and then the DNA sequence of the new antimicrobial peptide was obtained using DNA sequencing technology.-The biological properties of selected antibacterial peptides under different temperature,pH and enzyme tolerance,as well as inhibition spectrum and antibacterial potency were investigated.And a antimicrobial peptide with distinct biological property was picked out and named Naseain 9.-The amino acid sequence of Naseain 9 was determined to be VLTKKKPINTIYCGTVPCQGKKKRMGGLVKK by automatic Edman degradation sequencing technology,and the secondary structure of Naseain was analysized using the bioinformatics method.-DNA sequence of Naseain 9 suitable for expression in Bacillus subtilis host bacteria is optimized to be GTTCTKACAAAAAAAAAACCG-ATTAATACAATTTATTGCGGCACAGTTCCGTGCCAAGGCAAAAAAAAAAG AATGGGCGGCCTKGTTAAAAAA,based on Naseain 9 structure,combined with bioinformatics and preferred codon methods.-An efficient expression system BNAS for Naseain 9 was constructed based on the optimal combination of B.subtilis expression system components.And up to 89 mg of Naseain 9 could be obtained from 1 L fermentation supernatant at shake flask level.