The Expressions Of The Antibacterial Peptide Fowlicidin-3 In Different Expression Systems And Their Antimicrobial Efficiency

As an important component of innate immunological system, antibacterial peptides exhibit many characteristics such as wide antibiotic spectrum, high sensitivity to bacterials, special distinctive mechanism, higher thermostability and so on. To industrialize antibacterial peptides as a new antibacterial agents in veterinary clinic. The purpose of this study is to express Fowlicidin-3 by different expressions and detect their antimicrobial efficiency.1, Expression of Fowlicidin-3, a potential antibacterial agent with 27 amino acid, in Escherichia coli was explored. The DNA fragment encoding Fowlicidin-3a and Fowlicidin-3b was synthesized in light of the E.coli preferred codons by "partially overlapping primer-based PCR" method. The Fowlicidin-3 a and Fowlicidin-3b gene was fused with thioredoxin(Trx) gene to construct a recombinant plasmid p32C-F3a and p32TC-F3b. The fusion protein Trx-F3a and Trx-F3b was expressed.Then the soluble fusion protein was easily purified to near homogeneity by affinity chromatography using hexahistidine tag. Fowlicidin-3a and Fowlicidin-3b were effectively obtained by cleavage of the fusion protein with enterokinase and TEV protease. Antimicrobial activity assays demonstrated that the F3a and F3b had candidacidal activity.Integration of the key strategies for the expression of antimicrobial peptides(AMPs) such as codon usage bias,fusion partner and cleavage, would provide an efficient and facile platform for the production or study of AMPs.2, According to the amino acid sequences of Fowlicidin-3 as described in the antibacterial peptide database, using the preferential condon of P. pastoris, the antibacterial peptide Fowlicidin-3c gene 111 bp in length was designed and synthesized by "partially overlapping primer-based PCR" method. The antibacterial peptide gene was cloned into the pPICZa-A vector to construct the recombinant expression vector pPICZa-A-F3c. The linearized plasmid pPICZa-A-F3c was transformed into P.pastoris X-33 by electroporation. Under the control of the promoter AOX1, Fowlicidin-3c was expressed and secreted into supernatant. The concentration of the secreted peptides was 100μg/mL. The antimicrobial efficiency texts showed that Fowlicidin-3c had strong antibacterial abilities to E.coli DH5α.3, According to the amino acid sequences of Fowlicidin-3 as described in the antibacterial peptide database, we designed and expressed Fowlicidin-3 a、Fowlicidin-3b in Escherichia coli and Fowlicidin-3c in P. pastoris. The antimicrobial efficiency text of Fowlicidin-3a、Fowlicidin-3b and Fowlicidin-3c showed that Fowlicidin-3c exhibited the best antimicrobial activity, and Fowlicidin-3b was lower. The Minimal inhibitory concentration (MIC) of Fowlicidin-3a、Fowlicidin-3b、Fowlicidin-3c and Amp against E.coli DH5a were 2 μg/mL,2μg/mL,1μg/mL and 0.001μg/mL respectively.4, The therapeutic tests of antibacterial peptide Fowlicidin-3c, secretory expresion in recombinant yesat X33-p-F3c, in the E.coli k88 infected mice in vivo were studied in this experiment. After treated by different dosage of antibacterial peptide Fowlicidin-3c, the results showed that the mortality rate of the E.coli k88 infected mice decreased significantly in both antimicrobial peptide Fowlicidin-3c group and ciprofloxacin group compared to the E.coli k88 infected control group. The dosage of 100μg for each mouse shows the significant antibacteria effects in the E.coli k88 infected mice in vivo. The result from a decuple dose of antibacterial peptide Fowlicidin-3c treatment group also showed that it is safty to mice by oral administration.