Preparation Of Recombinant Antimicrobial Peptide Fowlicidin-1 And Its Antimicrobial Activity Analysis

Many of the antibiotics available today are secondary metabolites during microbe cultivation. Unfortunately, resistant strains have evolved, as they will almost inevitably do given sufficient time of exposure. A viable solution to this acquired resistance problemmay reside in new antibiotics, dissimilar to those that have lost their effectiveness. Antimicrobial peptide, one of the most important constituent components of host denfence system, is a kind of small peptides produced by inducing in organism. They are produced by all organisms, from plants and insects to human beings, as a major part of their immediately effective, nonspecific defences against infections. Although many demonstrate direct antimicrobial activity against bacteria, fungi, eukaryotic parasites and/or viruses, it has been established that antimicrobial peptides have a key modulatory role in the interface of innate and adaptive immunity. More recent evidence suggests that antimicrobial peptides are effective adjuvants, are synergistic with other immune effectors, initiate the adaptive response, support wound healing, induce or modulate of chemokine and cytokine production, alterate of gene expression in host cells, and inhibit of proinflammatory responses of host cells.In addition, the mechanisms of action are being unraveled, which support more effective implementation of derivatives of these endogenous peptides as therapeutic agents in overcoming infectious diseases. Chicken antimicrobial peptides Fowlicins are linear cation micromolecular polypeptides, their broad spectrum and salt-insensitive antibacterial activities against a range of Gram-negative and Gram-positive bacteria, including antibiotic-resistant strains, coupled with their potent LPS-neutralizing activity, make fowlicidins excellent candidates for novel antimicrobial and antisepsis agents. Chicken Fowlicidin-1 and its derivatives have efficient antimicrobial activity and other biological functions. Fowlicidin-18-26 is a cationic antimicrobial peptide composed of 19 amino acid residues derived from Fowlicidin-1. Fowlicidin-18-26 has more effective antimicrobial activity than native Fowlicidin-1 because of modification of amino acid sequence, and don't have rare amino acids and heterogenous chemical compositions, so it is one kind of great potential and secure antibiotics alternative. But according to our knowledge, there is no report on expression of Fowlicidin-18-26 in any type of expression system. The functional Fowlicidin-18-26 was successfully expressed in E.coli by gene engineering method in this experiment, which has insignificance in the development of basis of theory and practice to expressing Fowlicidin-18-26 and other small peptides, and also in the exploration of universal method and technology of gene engineering of peptides and antibacterial peptides research.The following results were obtained in this study:1. According to the biased codon used in E. coli, the gene sequence encoding active fragment of Fowlicidin-18-26 of mature Fowlicidin-1 was designed. The gene fragment of c-Fowlicidin-1 and d-Fowlidicidin-1 were obtained through PCR-based gene SOEing synthesis method.2. Multiple copies of c-Fowlidicidin-1 were inserted into the pET-32a prokaryotic expression vector using BglⅡand BamHⅠ(isocaudarner), constructed the fusion expression vectors c-p-Fowlicidin[1-6] with 1～6 copies c-Fowlidicidin-1 gene tandem in the same direction and transformed into competent cells of E.coli BL21 for fusion expression. The expression levels of c-Fowlidicidin-1 fusion proteins were examined by SDS-PAGE. The results showed that using pET-32a with one copy of the c-Fowlicidin-1 gene, the expressed level was relatively low, whereas no expression of multiple joinded genes was detectable in other vectors.3. The DNA sequence of d-Fowlidicidin-1 was inserted into the pET-32a vector, constructed the fusion expression vectors of p-d-Fowlidicidin and transformed into competent cells of E.coli BL21 for fusion expression. After IPTG induction, the results of SDS-PAGE and Western-bloting analysis showed that the d-Fowlicidin-1 fusion protein was successfully expressed. The highest productivity of the d-Fowlicidin-1 fusion protein was achieved by cultivating the E.coli in LB medium at 25°C, inducing the culture at the mid-exponential phase with 0.6mM IPTG, and collecting the broth after 11h expression. The expression level of soluble d-Fowlicidin-1 fusion protein exceeded 70% of the total soluble proteins under the induction of the optimized condition. The expression level of soluble d-Fowlicidin-1 fusion protein was 10 times higher in a new complex auto-inducing media introduced by our laboratory than in LB whose induction condition had been optimized The d-Fowlicidin-1 fusion protein was higher than 85% purity by Ni2+ chromatography purification as judged SDS-PAGE.Then the d-Fowlicidin-1 fusion protein was almost cleaved by recombinant enterokinase, and the antibacterial activity of fusion protein was tested by a liquid growth inhibition assay on E.coli,S.typhimurium,L.monocytogenes and S.aureus, which suggested the functional fowlicidin-1 has been achieved and first realized the heterologous expression of fowlicidin-1 by gene engineering method.