Bombyx Mori Antimicrobial Peptide Cm4 Fusion Expression In E. Coli And Its Anti-inflammatory Mechanism Study

Antibacterial peptides(AMPs)are cation polypeptide which play an important role in host defenses against microbial infection and are a key component of the ancient innate immune system。With more and more antibiotic resistant microorganisms emerging,antibiotic abusee accelerates this process.Antibacterial peptide's mechanism against target cells is remarkably different from that of traditional antibiotics.So they maybe have the potential value become a new generation anti-infection drug.The cecropin family of linear peptides without cysteine was found first in insects. ABP-CM4 is a small linearα-helics peptide that consists of 35 amino acids isolated from Chinese Bombyx mori by Zhang et al.ABP-CM4 has been proved to be a member of the cecropin family.ABP-CM4 kills bacteria,fungi and tumors by permeabilizing the cell membrane without being toxic to mammalian cells. To explore a new approach of expression of ABP-CM4 in E.coli,the gene of ABP-CM4 obtained by recursive PCR(rPCR)was cloned into the vector pET32a to construct a fusion expression plasmid.The fusion protein Trx-CM4 was expressed in soluble form,purified by Ni²⁺-chelating chromatography,and cleaved by formic acid to release recombinant CM4.Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC.The purified of recombinant peptide showed antimicrobial activities against E.coli K₁₂D₃₁,p.chrysogenum,A.niger and g.saubinetii.In order to efficacy of the antibacterial peptide(ABP-CM4)in binding Esherichia coli LPS in vitro.ABP-CM4 strongly bound to E.coli LPS,as proven by the limulus amoebocyte lysate(LAL)assay.Effects of LPS on the bactericidal action of the peptide were assessed.Flow cytometric analysis revealed that ABP-CM4 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells.Likewise,the inhibition of peptide to LPS-dependent cytokine induction was analyzed.Together these observations indicate that antibacterial peptide ABP-CM4 probably exert protective actions against endotoxin shock by blocking the binding of LPS to blocking of LPS to CD14⁺ cells.