| The increased bacterial resistance caused by the abuse of antibiotics had become a global public health problem. Therefore, seeking for a broad-spectrum, highly effective new generation of antimicrobial agents has become a serious problem over the world. In recent years, antimicrobial peptides as a novel antibacterial agent caused widespread concern in domestic and foreign researchers. Antimicrobial peptides(Antimicrobial peptides, AMPs), also known as peptide antibiotics, are a class of small molecule peptides with broad-spectrum antimicrobial activity against exogenous pathogens, and play an important role in natural immune defense system of the host. Currently, two kinds of antimicrobial peptidesit had been found in poultry, defensins and Cathelicidins. Research indicated that, the Cathelicidins antimicrobial peptides showed a strong and broad-spectrum activities on Gram-negative(G-) and gram-positive(G+) bacteria in vitro, and they also showed great potential. In the current study, we selected Silky Cathelicidins genes as the purpose genes to constructe eukaryotic expression plasmids to express the purpose proteins by Pichia pastoris(P. Pastoris) eukaryotic expression system. The expressed purpose proteins were combined with a variety of antibiotics to analyze their antibacterial effects, hoping to provide a substantial help to solve the problem of antibiotic resistance, and lay the foundation for further studies in combination with antibiotics.To amplify the Cathelicidin-1,-2,-3 genes, three pairs of primers were designed according to the Cathelicidin-1,-2,-3 genes sequences(GeneBank accession number: FJ938357、FJ938358、FJ938359). EcoR I and Xba I restriction enzyme sites were introduced at the respective 5′-termini. The RNA of Silky were collected from its leg bine marrow and then amplify the Cathelicidin-1,-2,-3 genes through RT-PCR. The RT-PCR products were cloned into the pMD18-T vector, and the resultant plasmids were confirmed by sequencing. The resultant plasmids pMD18-T-Cathelicidin-1,-2,-3 were digested with EcoR I and Xba I, and cloned into the pPICZα-A vector, which was digested with the same restriction enzymes. The recombinant plasmids were confirmed by restriction digestion and sequencing. The recombinant plasmids pPICZα-A-Cath L1,L2,L3 and plasmid pPICZα-A were digested with Sac I to obtain the linearized plasmids. Then the linearized plasmids were transformed into competent P. Pastoris cells by Electroporation, respectively. Correct positive transformants were by PCR. The Mut+ phenotype and high copies selecting were going with the correct positive transformants to obtain the final strain, which can use the methanol.The positive recombinant yeast strains were induced by methanol to express the protein, with adding methanol(final concentration of 1.0%, 2.0%) per 24 h and collecting 1 m L cell culture supernatants, until to 96 h. Then the expressed proteins in the cell culture supernatants was purified with DOC-TCA, and verified by Tricine-SDS-PAGE. The results showed that three single protein bands with apparent molecular weight of 7.6 KD, 8.1 KD, 7.6 KD were obtained after the purification procedure, which was consistent with the expected results. In addition, the results of HuaDa company showed that the expressed proteins were correct. Notably, the results showed that the positive transformants in 1.0%, 2.0% methanol had highest protein expression at 72 h, and the protein expression in 2.0% methanol was higher than in 1.0% methanol.Through the orifice 96 micro-dilution method is used to determine the bacteriostatic effect of expression products.Test results show that the three kinds of Cathelicidins are Escherichia coli, Pseudomonas aeruginosa, Singular proteus showed different degrees of bacteriostatic effect, the MIC value between 2-64 μg/mL, and antibacterial peptide Cathelicidin-1 for E.coli 1505 has obvious antibacterial effect, the MIC value of 2 μg/m L, is better than that of ciprofloxacin(MIC=4 μg/mL), ampicillin(MIC=32 μg/m L), ampicillin(MIC=32μg/mL), cephalosporins he organism(MIC=32 μg/m L) and gentamycin(MIC=16 μg/m L), levofloxacin(MIC=8 μg/mL).Three kinds of antibacterial peptide cathelicidin and amoxicillin, amikacin, archie, and other 11 kinds of antibiotics and research on E.coli、P.aeruginosa、S.pullorum、P.multocida、Micrococcus luteus 、S.aureus strains of antibacterial effect, the bacteriostatic effect of antibiotics as control.United bacteriostasis results show that for different strains of antibiotic and antimicrobial peptides have synergy effect, also have antagonism effect.Among them, in the joint antibacterial effect for pseudomonas aeruginosa, amikacin has no antibacterial effect when used alone, but and restructuring of antimicrobial peptides Cathelicidin-1,-2,-3, when used, showed a very significant bacteriostatic effect(P < 0.01); For 1507, 0914 strains of E.coli, cefoxitin no antibacterial effect when used alone, but and restructuring of antimicrobial peptides cathelicidin-1,-2,-3, when used, show that the bacteriostatic effect, and synergy effect is extremely significant(P < 0.01). But for 1504 strains of E.coli, ofloxacin bacteriostatic effect when it is used alone, but and restructuring of antimicrobial peptides cathelicidin-1,-2,-3, when used, expression is no antimicrobial effect, antagonism effect is very significant(P < 0.01). The results showed that the poultry cathelicidin antimicrobial peptides and the interaction between different antibiotics, the combination of certain showed obvious bacteriostatic potential synergy. According to different bacterias, exploration has obvious antibacterial effect of cathelicidin antimicrobial peptide antibiotics and prescription is a feasible way to reduce the antibiotic usage. |
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