The Cloning, Expression And Anti-cancer Antibacterial Mechanism Of The Antibacterial Peptides Scopolin1 And Scolopin2 Were Studied

As a traditional Chinese herbal medicine,the venoms of centipede play an important role in the innate immune system.AMP-scolopin 2 and AMP-scolopin 1 were identified from centipede venoms of Scolopendra subspinipes mutilans.Both scolopin 2 and scolopin 1 have broad-spectrum activities against bacteria,fungi.As cancer is the biggest threat to human health,looking for efficient and specific killing malignant cells become the main resource of the new anti-cancer research.Some cationic antimicrobial peptides exhibited cytotoxic activity against cancer cells,becoming an important class of antimicrobial cancer resources,we further investigated the anticancer activity of scolopin 2.We first constructed an efficient system for the expression and purification of scolopin 2 and scolopin 1 in E.coli by the pSUMO expression vector.This thesis contains four chapters:1.Some advances in the studies of antimicrobial peptides and anticancer mechanism.2.The research progress of the centipede Scolopendra subspinipes mutilans.3.We first report here the application of small ubiquitin-related modifier(SUMO)fusion technology to the expression and purification of scolopin 2.The antimicrobial activity of purified recombinant AMP-scolopin 2 was tested by an inhibition zone assay.To assay the anticancer activity and side effects,hemolytic and apoptosis assays were employed.Results demonstrated that AMP-scolopin 2 had cytotoxic and apoptotic activity in K562 leukemia cells and HepG2 liver carcinoma cells,while it had only a minimal effect on erythrocytes and normal HEK293 cells.In summary,the present study indicates that scolopin 2 can inhibit the proliferation of tumor cells in vitro through mitochondrial caspase-dependent pathway of apoptosis.4.Antimicrobial peptide scolopin 1(AMP-scolopin 1)is a small cationic peptide identified from centipede venoms of Scolopendra subspinipes mutilans.We first report here the application of small ubiquitin-related modifier(SUMO)fusion technology to the expression and purification of cationic antimicrobial peptide AMP-scolopin 1.The fusion protein expressed in a soluble form was purified to a purity of 95%by Ni-IDA chromatography.After the SUMO-scolopin 1 fusion protein was cleaved by SUMO protease at 30?for 1 h,the cleaved sample was reapplied to a Ni-IDA.The recombinant scolopinl had similar antimicrobial properties to the synthetic scolopin 1 by an inhibition zone assay,using Gram-negative bacteria E.coli K12D31 as test bacteria.The minimal inhibitory concentration(MIC)was defined as the lowest concentration of peptide at which there was no change in optical density.The SEM experiments indicated that scolopin 1 progressively destroyed the bacterial envelope with the increase of concentration and caused the leakage of cell contents which led to cell death by binding to cell membranes of E.coli K12D31.