High - Level Expression, Remodeling And Anti - Pseudomonas Aeruginosa In Antimicrobial Peptide

With the abusive application of a large number of antibiotics, antibiotics are subjected to certain constraints for bacterial resistance and screening bottleneck of new antibiotic. In recent years, antimicrobial peptides (AMPs) had became the most ideal alternative medicine for the unique mechanism of action and molecular characteristics. Antimicrobial peptide CM4was isolated from the hemolymph of the silkworm Bombyx mori by our laboratory. CM4which is a typical amphipathic a-helix peptide has broad antibacterial effect, and no toxicity to normal cells, thus having good application prospect. In order to expand the application of the CM4, we adopted prokaryotic expression system to optimize the CM4expression and purification. And we improved its antibacterial activity by CM4transformation or modification, and then we study the antimicrobial mechanism of CM4to Pseudomonas aeruginosa.The research of the concrete is divided into5parts:1The expression and purification of AMP-CM4Earlier attempts to produce recombinant ABP-CM4in our laboratory using fusion technology had some disadvantages such as low levels of cleavage efficiency or low yield. We adopted the SUMO (Small Ubiquitin-like Modifier) fusion protein expression system developed by USA LifeSensors company. After subcloning of the cDNA of CM4,2CM4and3CM4into pSUMO vectors, optimizing expression conditions, SUMO protease cleavage, Ni column purification, antibacterial peptide CM4was obtained with the purity of95%, and the recombinant antibacterial CM4had the same antibacterial activity with the chemical synthesis. This is the first report to express cecropin of AMP-CM4with SUMO fusion, which not only shorten the process time also improves the expression level compared with previous expression, and the final yield of CM4was24mg/l.2The molecular modification of AMP-CM4In order to improve the antibacterial activity of AMP-CM4, the CM4structure was modified including forming new hybrid peptides from CM4, Magainin Ⅱ and Melittin, and C-terminal asparagine modification of CM4. Through the SUMO expression system, the activity of the new hybrid peptides and mutant was lower than the parent CM4, but CM4N of C-terminal asparagine modification had stronger activity than the parent CM4. This is the first research of AMP-CM4molecular modification, and we obtained a stronger antibacterial peptide CM4N.3The stability optimization of AM PCM4NIn order to improve the protease sensitive and stability, we prepared the supramolecular compound (3-CD/CM4N by using the conventional solution. Then we analysed characters of the β-CD/CM4N by ultraviolet visible spectroscopy, DSC (differential scanning calorimetry) analysis and antibacterial activity analysis. This is the first study of P-CD to improve the stability of AMP-CM4N.4The antimicrobial mechanism of CM4N to Pseudomonas aeruginosaWe studied the the antimicrobial mechanism of CM4N to Pseudomonas aeruginosa. Through the study of CM4N bacteriostatic concentration and bactericidal time curve, after determining the minimum inhibitory concentration of CM4N (20μM), the scanning electron microscope and transmission electron microscopic observation of the cell membrane of Pseudomonas aeruginosa by dealing with CM4N, and the interaction between the CM4N and the genomic DNA or RNA, we first report the antimicrobial mechanism of CM4to Pseudomonas aeruginosa by forming transmembrane pores on the biofilm and nucleic acids binding at the molecular and the cellular level.5The protection of AMP-CM4N on mice with sepsisIn vitro studies have found antibacterial peptide CM4N can effectively kill Pseudomonas aeruginosa. In order to study the killing effect of CM4N on Pseudomonas aeruginosa in vivo, we constructed a mouse model with Pseudomonas aeruginosa infection. After observing the signs change of animals, survival rate, changes of tissue pathological, we evaluated the protection of AMP-CM4N on mice with Pseudomonas aeruginosa infection preliminary. In order to extend the half-life in vivo and the decrease of serum on CM4N activity, the supramolecular compound (3-CD/CM4N was used.