Construction And Bioactivity Of Eukaryotic Recombiant Plasmid Of Novel Fusion Gene Of Antibacterial Peptide

Antibacterical peptide (ABP) is a kind of small molecule polypeptide encoded by special cells from animals and plants, which can resist against microorganism and eliminate mutant cells, also named antimicrobial peptide (AMP) and peptide antibiotics. Nowadays, ABP have been attracting much concerns for its strong inhibitory effect on drug resistant bacteria. Based on the information about the relation of structure and fuction of antibacterial pepetides, the molecular design and reconrtruct of antimicrobial peptides is a new effective way to develop novel medicines against infection.In order to develop novel safe and effective molecules against animal infection, the experiment was conducted to subclone the fusion gene of novel antibacterial peptide, ECD, which had been contructed in our lab, into eukaryotic expression plasmid, VR1020, and express it in HEK 293 eukaryotic cells. The cDNA of ECD was amplified by PCR from pGECD and digested with BamHI/BglII double digestion, and then was ligated with the VR1020 digested with the same endonucleases and dephosphated by calf intestine alkine phosphatase. The recombinant paslmids was screened out by incubating the transformed E. coli DH5αcells in LB containing Kanamacin, and identified by plasmid PCR and BamHI/BglII double digestion. The correct recombinant was confirmed by DNA sequencing and designed as VR-ECD.The HEK293 eukaryotic cells was transfected by the purified VR-ECD which was respectively packed with JetPEI^TM Cationic polymer transfection reagent and chitosan nanoparticles (CNP) that was prepared in our lab through ionic across linkage method. 24, 48, 72hs after transfection, the total RNA was isolated from from the transfected eukaryotic cells, and the mRNA transcriped from VR-ECD was detected by RT-PCR. The results showed that the mRNA encoded by ECD cDNA was correctly expressed in the transfected cells; and the supernatants of transfected cells culture medium were utilized to analyze the antibacterial activity against five strains of bacteria. The supernatant from the VR-ECD transfected cells showed significant inhibition effect against the growth of E.coli, ETEC, Pseudomonas aeruginosa, Streptococcus pneumoniae and Staphylococcus aureus in comparison with those of negative and blank controls (P<0.05) . These suggested the recombinant eukaryotic plasmid, VR-ECD, could express the fusion defensin with strong antibacterial effect to suppress the growth of bacteria in vitro, indicating that the recombinant VR-ECD could be further developed into a novel effective antimicrobial molecule to prevent and control the infections in animals in the future.