Studies On High Level Expression And Purification Of Antimicrobial Peptide Cec Md From Musca Domestica In E.coli And Activities Of Its Expression Products

As the extensive application even the abuse of antibiotics, especially antimicrobial growth promoters (AGPs) in animal husbandry over the recent years, more and more antibiotic-resistant bacteria have emerged, and even many serious problems involving food safety and environmental pollution were triggered accordingly, which has ceaselessly plagued the public though antibiotic has been thought as the booster of animal production. Antibiotic abuse in animal husbandry decreases the quality of animal products and threats the health status of general public, which not only jeopardizes the public health security, does detrimental to operation of industry chain of animal husbandry, but also weakens competitiveness of animal products of our country in global market. Antimicrobial peptides (AMPs) are kinds of cationic peptides with broad antimicrobial spectrum and they are essential components of innate immune system. At present, many study fields such as biology, medicine, pharmacy, and agronomy focused AMP for its high-performance and unique action mechanisms. AMPs possess such several merits as non-drug resistance, non-residual, high temperature resistance and high specificity on target cells, which make itself as the substitute of traditional antibiotics in feed industry. Therefore, further doing the basic researches on antimicrobial peptide is significance to improve quality of animal products, enhance performance, increase resistant ability to diseases, even to promote development of the entire animal husbandry. Some works were conducted as following:(1) Cloning and bioinformic analysis of gene of Cec Md from Musca domestica larvae. Total RNAs were isolated from Musca domestica larvae with or without lipopolysaccharide (LPS) challenging and cDNA encoding the open reading frame (ORF) of Cec Md were amplified by RT-PCR using primers designed according to the known sequence of Musca domestica Cecropin (GenBank: DQ232774). Gene fragments of Cec Md were ligated into pMD-18T vector and, then were identified by PCR, double restriction enzyme digestion and sequencing. Bioinformic analysis on fragments of Cec Md gene and its deduced amino acids were completed. Results indicated that gene of Cec Md was successfully cloned from Musca domestica larvae challenged with LPS but failed to obtain it from the maggots without LPS inducements. The coding region of Cec Md was 192 bp in length which encoded a polypeptide consisted of 63 amino acid residues which consisted of 8 strong basic (+) amino acids, 4 strong acidic (-) amino acids, 30 hydrophobic amino acids and 14 polar amino acids. the most likely cleavage site was between residue 23 and 24, which demonstrated that the part of 1～23 amino acids in Cec Md was signal peptide and of 24～63 amino acids was mature peptide. Results of prediction on three-dimensional (3-D) structure models showed that the complete peptide, a mature peptide plus signaling peptide, of Cec Md contained four helixes whereas the mature peptide only possessed two, both of which were no disulfide bonds. Analysis of homology of sequence indicated that the homologous rates of amino acid sequences of cecropins between Musca domestica and Drosophila were higher than those of other species, reaching 84.1%.(2) Expression of Cec Md gene in E.Coli and purification of its expression products. Fragment of Cec Md gene from pMD18-T/Cec Md with double digestion were cloned into expression vetor pET32a (+) or pGEX-6P-1 respectively and then identified by PCR, digestion of double restriction enzymes and DNA sequencing. Results showed that two recombinant expression vectors pET32a (+)/Cec Md or pGEX-6P-1/Cec Md were constructed successfully and then the recombinants were transformed into E.coli BL21 (DE3) with induction of IPTG and, finally identified the expression level of target protein using SDS-PAGE. Results demonstrated that bands of protein from pET32a (+)/Cec Md were not detected whereas pGEX-6P-1/Cec Md expressed its protein in E.coli BL21 (DE3) with the identification of SDS-PAGE and Western blot. It indicated that genetic engineering strains was constructed and the proteins were highly expressed as inclusion body. Fusion protein was purified using affinity chromatography column High-Affinity GST Resin, and digested with PreScissionTM Protease. Rtesults indicated that concentration of Cec Md fusion protein in samples was 336.83mg/mL , and the yield rate of Cec Md expressed in E.Coli. was 650mg from per liter medium.(3) Optimization of expression conditions of recombinants. Growth curves of recombinant pGEX-6p-1/Cec Md/BL21 (DE3) in various cultures were assayed and the results showed that TB medium was the optimum one for the growth of the bacteria. After induction of IPTG, growth curves of E.Coli were altered and density of recombinant cells was lower than those of non-recombinant ones, which indicated that Cec Md has antibacterial activity. Optimization of expression conditions of fusion protein were preliminary explored and the results showed that the optimum combination for fermentation of the cells were: 1) culture: TB; 2) concentration of IPTG: 0.8mmol/L; 3) time of induction: 6h; 4) temperature: 37℃. Optimum expression conditions of fusion protein were determined in this work, which provided experimental basis for the high-density culture of recombinants strain pGEX-6p-1/Cec Md/BL21 (DE3) in fermentation system.(4) Studies on activity of Cec Md. Results indicated that both Cec Md and Cec Md fusion protein (without digestion of PreScissionTM Protease) inhibited the growth of Escherichia coli, Salmonella gallinarum and Streptococcus dysgalactiae. Inhibition zones among Cec Md, Cec Md fusion protein and ampicillin were in same size. Minimal inhibitory concentration (MIC) of Cec Md on Escherichia coli and Streptococcus dysgalactiae were 50% lower than those of ampicillin, and Cec Md at concentration of 5.86μmol/L inhibited growing of Streptococcus dysgalactiae while ampicillin did not show antibacterial activity on this bacteria until its concentration reached 750μmol/L. Evaluation of thermal stability indicated that Cec Md possessed well thermal stability and its structure was not destroyed and, accordingly its activity was maintained in a high temperature condition. Results demonstrated that lymphocyte transformation rates in peripheral blood in SD rats and Sanhuang chickens were increased by Cec Md, which demonstrated that this peptide played an active part in regulatory function in cellular immunity.