Heterologous Expression Of Chicken AvBD7Mature Peptide In Escherichia Coli And Antimicrobial Activity Analysis

AvBD7mature peptide is a42-amino acid residue β-defensin produced by thechicken, belong to the large family of cationic antimicrobial peptides(CAMPs), whichis a important component of innate and adaptive immunity. It is a capable of killing abroad spectrum of pathogens, and microbial resistance may occur with lowerprobability than observed with ‘conventional’ anti-infective agent. Towardslarge-scale production of the defensin is current and future field of research.In order to study the expression in E. coli system and antimicrobial activity ofAvBD7mature peptide in vitro, we constructed an engineered E. coli strain containschicken AvBD7gene encoding AvBD7mature peptide published previously bygenetic engineering technique. Based on the gene sequence, we synthesized theAvBD7gene, cloned into pGEX-6p-1prokaryotic expression vector and subsequentlytransformed into the E. coli BL21(DE3). The E. coli strain contains the expressionvector was induced in the most optimal conditions and the fusion protein GST-AvBD7was purified by affinity chromatograph, then Prescission protease was used toremove GST-label to form AvBD7mature peptide. The Purification of chickenAvBD7mature peptide was determined by MALDI-TOF-MS displayed notableantimicrobial activities. Specific contents and results are as follows:1. Construction of recombinantant E. coli expressing chicken AvBD7maturepeptideBased on the gene sequence of chicken β-defense in GenBank(registration numberDQ858344), we synthesized the AvBD7mature peptide gene sequence, cloned intopGEX-6p-1prokaryotic expression vector and subsequently transformed into the E.coli BL21(DE3). PCR amplification the chicken AvBD7mature peptide gene throughthe design of primers P1-sense primer (EcoR I restriction sites) and P2-anti-senseprimer (Sal I restriction sites), cloned into pGEX-6p-1prokaryotic expression vectorand subsequently transformed into the E. coli BL21(DE3) after double digestion.2. Expression, purification and identification of chicken AvBD7mature peptideThe sequencing of E. coli BL21(DE3)/pGEX-6P-1-AvBD7inoculated in LB liquidmedium (Amp final concentration was100μg/mL), the fusion protein GST-AvBD7was induced by IPTG, SDS-PAGE detection molecular weight is about31kD. Toascertain the best expression of the soluble fusion proteins according to explore theconditions of induction in different temperature, time, and IPTG concentrations. A high level of expression of the recombinant proteins in supernatant were obtainedafter induction for24h with0.1mM IPTG at16°C(120r/m). The fusion proteinGST-AvBD7was purified by affinity chromatograph, ultrafiltration, and C18ZipTipchromatography then Prescission protease was used to remove GST-label to formAvBD7mature peptide. The molecular weight of purified AvBD7mature peptide wasidentified as5516Da by MALDI-TOF-MS shown that formed two disulphide bridgesin accordance with the predicted molecular weight.3. Chicken AvBD7mature peptide antimicrobial activity analysisTo investigate the antimicrobial activity of AvBD7mature peptide using indicatorstrains to perform by the agar well diffusion assay. The results showed that theantimicrobial activity of AvBD7mature peptide against Micrococcus flavus (NCIB8166), Staphylococcus aureus (ATCC25923), E.faecalis (ATCC29212), E. coli(CMCC44102), but no inhibition to Candida albican (ATCC10231) and Candidaalbican (ATCC90029). The concentration of samples were determined byMALDI-TOF MS that were measured by Bradford’s method using the BCA Kit. Theminimum inhibitory concentration (MIC) of AvBD7mature peptide against variousindicator strains by a method slightly modified from that of Levengood. The MICvalues were16.875,67.5,67.5,135mg/L individually correspond to Micrococcusflavus (NCIB8166), Staphylococcus aureus (ATCC25923), E. faecalis (ATCC29212), E. coli (CMCC44102). In addition, the high concentrations(>540mg/L) ofAvBD7mature peptide not exhibited antimicrobial activity against Candida albican(ATCC10231) and Candida albican (ATCC90029). Therefore, eventually obtainingthe chicken AvBD7mature peptides with antimicrobial activity only to some certainindicator bacteria in vitro.In summary, We successfully constructed the recombinantant engineered E. colistrain expressing pGEX-6P-1-AvBD7and find out the optimal induced conditions.The molecular weight of purified AvBD7mature peptide was monitored byMALDI-TOF-MS and antimicrobial activity was tested by the agar well diffusionassay. Besides2-fold dilution method was used to determine the minimum inhibitoryconcentration of AvBD7mature peptide. It is very important to develop noveldefensin feed additives, which will enrich our knowledge about defensin.