Eukaryotic Expression Of Cecropin P2, Cecropin A And The Fusion Protein Lysozyme-Cecropin A, And Studies On The Antimicrobial Activity

With the rapid rise in the emergence of bacterial strains resistance to antibiotics, find a new type ofantimicrobial agent to replace the antibiotics is imminent. Antimicrobial peptides (AMPs), which couldfind in a wide variety of species and came from animal immune system are small molecular peptidesand an important part of the innate immune system. AMPs is one kind of defensive peptides, whichresist to the invasion of exogenous pathogens, having broad-spectrum antibacterial activity (likeanti-bacteria, viruses, fungi, parasites and tumors)and difficult to generate resistance. In recent years,AMPs, through its unique mechanism of action and biological activity, has become a research hotspot inmany fields, and has a broad prospect in application. Cecropins is a kind of cationic AMPs which hasgood antibacterial activity to Gram-negative and Gram-positive bacteria, and has large potentiality indrug development.This study related two AMPs that are Cecropin A and Ascaris suum Cecropin P2.Cecropin A, is one of the first discovered AMPs which has35amino acid residues and has the typicalα-helix structure of Cecropins, has broad-spectrum antibacterial activity. Cecropin P2is a polypeptideisolated form Ascaris suum, which molecular weight is about4.2kDa and the amphiphilic structure issimilar to the insect cecropins. Lysozyme is a kind of small molecule substances from organisms, whichhave thermal stability, high efficiency, not easy produce drug resistance and other characteristics likeAMPs.In this study, the AMPs coding genes cecropin P2and cecropin A were got by overlap PCR according tothe coding sequence of cecropin P2and cecropin A from GenBank. Meanwhile, a fusion geneLysozyme-Cecropin was got by connected the coding gene of lysozyme and cecropin A together. Inorder to construct secretory expression plasmids of Cecropin P2, Cecropin A and Lysozyme-Cecropin A,three target genes were inserted into the multiple cloning sites of pPIC9K (secretory expression vectorof yeast).Three expression plasmids which were identified by PCR and restriction enzyme digestionwere linearized by the restriction enzyme of Sal I and electro transformed the Pichia pastorios GS115express bacterium. Transformants with multi-copy genes in Mut+type after the transformants werescreened by Minimal Dextrose medium (MD), Minimal Methanol medium (MM) and the antibiotic ofG418. The results of PCR identification demonstrated that Cecropin P2, Cecropin A andLysozyme-Cecropin A were inserted into the chromosomes of express bacterium. The secretoryexpression of Cecropin P2, Cecropin A and Lysozyme-Cecropin A were performed under the inductionof methanol.The results of Tris tricine-SDS-PAGE and SDS-PAGE indicated that the three targetproteins were secreted into supernatant, and demonstrated that the three recombinant yeast can secretedexpress target proteins respectively.In order to test the antibacterial activity and verify the function of the recombinant AMPs such asCecropin P2, cecropin A, and the fusion protein Lysozyme-Cecropin A, this study used the agardiffusion method and microdilution method to determine antibacterial activity and minimum inhibitory concentration (MIC) of the three recombinanted proteins to Escherichia coli(E.coli) and Staphylococcusaureus (S.aureus); and the cytotoxicity of recombinant proteins was detected by co-cultured the AMPsand cells such as Madin-Darby Bovine Kidney cells(MDBK), Chinese Hamster Ovary cells (CHO) anddendritic cells (DC); and the antimicrobial activity of recombinanted proteins against Chlamydophilaabortus was detected via the medium of chick embryo. The results demonstrated that the recombinantCecropin P2, cecropin A, and the fusion protein Lysozyme-Cecropin A have not cytotoxicity to MDBK,CHO, and DC. Meanwhile, the results also proved that the recombinant proteins have antimicrobialactivity to E.coli, S.aureus and Chlamydophila abortus.In summary, recombinant AMPs which have biological activity were obtained by Pichia pastorisexpression system, and this study will provide necessary experimental data for the application of AMPsand the development of new AMP drugs.