| Chicken β-defensin is a kind of cysteine-rich small peptide, which has become aresearch hot due to a broad spectrum of resistance to bacteria, a special resistancemechanism, no residue and so on, will play a greater role in the prospect of the futureapplications of animal husbandry. Chicken avian beta-defensin-2(AvBD2) is an importantchicken antimicrobial peptide. It widely distributed in the bone marrow and lung. It hasbeen proved that AvBD2has strong antimicrobial function.Based on the sequence of AvBD2mature peptide gene, considered Escherichia colicodon bias, a new AvBD2mature peptide gene was designed and synthesized. The cloningvectors and expression vectors were constructed. The objective fusion protein was inducedwith IPTG and purified with GST resin. Optimal levels of GST-AvBD2were obtained byoptimizing the concentration of IPTG and the induction time. The antibacterial activity ofpurified product was investigated. The results were showed as followed:1Design and Synthesis of AvBD2mature peptide geneThis experiment based on the published sequence of AvBD2mature peptide inGenbank, considered Escherichia coli (E. coli) codon bias, designed and synthesized anew chicken beta-defensin-2mature peptide gene (AvBD2op) by selecting high-usagecodons in E.coli. The restriction enzyme cutting sites of BamHI and EcoRI were added ateach end of the gene sequence. The chicken beta-defensin-2mature peptide gene (AvBD2)was synthesized and the restriction sites NotI and EcoRI were added at each end of thegene sequence. The cloning vectors of pMD18-Tsimple-AvBD2op and pMD18-Tsimple-AvBD2were constructed.2Expression and purification of AvBD2fusion proteinThe AvBD2mature peptide gene and codon optimized mature peptide AvBD2op genewere double digested and inserted into the pGEX-6P-1vector which was also doubledigested. The recombinant vectors named pGEX-6p-1-AvBD2and pGEX-6p-1-AvBD2opwere transformed into E.coli BL21cells.The recombinant plasmids were isolated andpurified after enlargement culturing, and verified by PCR and gene sequence analysis. Theblast result displayed that the homology of mature peptide was100%. The positive cloneswere cultured to logarithmic phase and induced to express target protein by addition ofIPTG. Expression products with molecular weight of approximately30kD were examined by SDS-PAGE. The soluble fusion protein was best expressed at20℃by0.5mmol/LIPTG after5h. The expressed fusion proteins were purified with GST resin, and thepurified protein concentration was determined by BCA Protein Assay Kit. The resultshowed that the expression level was remarkably improved by using E.coli bias codons.3Antibacterial activity of AvBD2fusion proteinThe antimicrobial activity of the fusion protein was determined by monlayer agar platediffusion method, which exhibited antimicrobial activity against Enterococcus faecalis(ATCC29212), Salmonella pullorum (S2), Escherichia coli (CMCC44102), Staphylococcusaureus (ATCC25923). The minimal inhibitory concentration (MIC) of the fusion proteinagainst above bacteria ranged from31.25to125μg/mL. By the observation under thetransmission electron microscope，it was founded that the fusion protein made the celldistorted, broken and cell components releasing, which tumed the cell into irregularresidues.In summary, we synthesized AvBD2mature peptide codon optimized gene, andconstructed its expression vectors. The analysis of the soluble fraction and the cell debrispellet suggested that GST-AvBD2was induced at20℃using0.5mmol/L IPTG, themajority of the fusion protein is soluble. The bacteriostasis study revealed that purifiedfusion protein had antibacterial activity by damaged the bacterial structure. This studyestablished the foundation for production and application of new preparations of thechicken beta-defensin-2genetic engineering. |
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