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Studies on High Level Expression and Purification of Antimicrobial Peptide Cec Md from Musca Domestica in E.Coli and Activities of Its Expression Products

Author: ZhaoPingSen
Tutor: JiangNing
School: Heilongjiang Bayi Agricultural University
Course: Animal Nutrition and Feed Science
Keywords: Musca domestica Cec Md E. coli Highly expressed Protein Purification Expression optimization Activity Detection
CLC: S816
Type: Master's thesis
Year: 2009
Downloads: 30
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Abstract

Widely used antibiotics in animal husbandry and even abuse, reduce the quality of animal products, and a threat to the health interests of consumers. This not only harmful to the public health security of our country, to undermine the normal operation of the livestock industry chain, and greatly weakened the competitiveness of animal products in the international market. Antimicrobial peptides (Antimicrobial peptides, AMPs) for a class of cationic small peptide organisms are important components of the innate immune system, with a broad spectrum of the Hang bacteria activity. AMPs its unique biological activity and mechanism of action, has become a hotspot of biology, medicine and agriculture fields. AMPs have no pathogen resistance, no residue, high temperature, and has the advantage of highly specific target cells, will be the ideal substitute of traditional antibiotics in the feed industry. Therefore deepen the antimicrobial peptides of the development of basic research and its family of products is of great significance to improve the quality of livestock and poultry products, improve livestock production performance and disease resistance and even promote the development of animal husbandry. The research is mainly carried out exploration of the following aspects: (1) Cec Md gene cloning and bioinformatics analysis. Housefly lipopolysaccharide (LPS)-induced and non-induced extraction of total RNA, according to the housefly cecropin gene (GenBank: DQ 232774) specific primers were designed using RT-PCR amplified coding Cec Md open reading frame (ORF) cDNA sequence, and cloned into pMD18-T vector, after PCR, double digestion and sequencing, this gene and its deduced amino acid sequence bioinformatics analysis system. The results showed that amplified from LPS-induced Housefly Musca domestica cecropin gene, but never induced insects were unable to obtain this gene; cloned housefly Cec Md gene size 192 bp, encoding 63 amino acids a polypeptide consisting of residues, MW 6739.94, 8 wherein the strongly basic amino acids, strongly acidic amino acids 4, 30 hydrophobic amino acids, polar amino acid 14; Cec MD most likely cleavage site between the A23 and G24 Step N-terminal amino acids 1 to 23 CEC Md signal peptide, C-terminal 24 to 63 amino acids constituting the mature peptide; 3-D ??structure prediction results show that the CEC Md mature peptide contains two helical structure, excluding disulfide; CEC Md homology with Drosophila cecropins significantly higher than that of other insects, more than 81.0%, the highest of 84.1%. (2) Cec Md gene in E.coli expression product was purified. Constructed pMD18-T/Cec Md after double digestion to obtain a linear target gene fragment directed its recombinant expression vector pET32a () and pGEX-6P-1 by PCR, restriction analysis and DNA sequencing Analysis. Determine the success of the recombinant expression vector pET32a () / Cec Md and pGEX-6P-1/Cec Md transformed into E. coli BL21 (DE3), IPTG induced expression of the detection of the expression of the target protein, SDS-PAGE. The results show that not detected in SDS-PAGE film pET32a. () / Cec Md recombinant vector protein strip; detected by SDS-PAGE and Western blot, The proof pGEX-6P-1/Cec Md obtained in the BL21 (DE3) abundantly expressed successfully established pGEX-6P-1/Cec Md/BL21 (DE3) recombinant genetic engineering strains; recombinant protein form of inclusion bodies, the latter with a High-Affinity GST Resin affinity chromatography purified, and then use PreScissionTM Protease of the fusion protein to be cut, Cec Md obtain a purified protein; Determination draw the present protein sample Cec Md fusion protein containing 336.83mg/mL, Cec Md expressed in E. coli BL21 (DE3), the yield was 650mg / L culture solution. (3) Restructuring engineering bacteria to express the optimization of the conditions. Determined the growth curve of recombinant genetic engineering bacteria pGEX-6p-1/Cec Md/BL21 (DE3) in a different medium. The results show that: TB medium most suitable for the growth of recombinant genetic engineering bacteria; after induction with IPTG, cell growth curve change, antimicrobial peptide growth of recombinant bacteria density lower than the control group (non-recombinant genetic engineering bacteria). Found that expression of antimicrobial peptides can reduce the growth of the host bacteria density, this effect from the side reaction Cec Md antibacterial activity of the fusion protein; fusion through Cec Md preliminary exploration of the best protein expression conditions, to determine the optimum fermentation conditions were : ① medium: TB medium; ② IPTG was concentration: 0.8mmol / L; (3) induction time: 6h; the ④ temperature: 37 ° C. Cec Md, finalized the pGEX-6p-1/Cec Md/BL21 (DE3) strain highly expressed in the conditions of high expression to its high density culture in bioreactor provides an experimental basis. (4) Musca domestica Cec Md activity. Cec Md and Cec Md fusion protein (Cec Md fusion protein) inhibited E. coli, Salmonella and Streptococcus dysgalactiae cut without PreScissionTM Protease growth, both inhibition zone size is similar, with the suppression of ampicillin. bacterial activity close; Cec Md three strains of E. coli, Salmonella and dysgalactiae Streptococcus minimum inhibitory concentration (MIC) were lower than the ampicillin group Cec Md minimum inhibitory concentration (MIC) of E. coli and Salmonella gallinarum ampicillin group 50%, CEC MD at a concentration of 5.86μmol / L, on the growth of Streptococcus dysgalactiae inhibition, but ampicillin only concentration GT; 750μmol / L appears when the antibacterial activity. The thermal stability testing Cec Md better thermal stability, high temperature 20min not destroy the structure and antibacterial activity of Cec Md. Cec Md can significantly improve laboratory animals lymphocyte transformation rate Cec Md positive role in promoting cellular immune function by exploring the impact of Cec Md SD rats and Sanhuang, lymphocyte transformation rate, .

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