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PAMP37-PR39 fused antibacterial peptide and preparation method thereof

An antibacterial peptide and antibacterial immunity technology, applied in the field of PAMP37-PR39 fusion antibacterial peptide and its preparation, can solve the problems of increasing disease treatment difficulty, high cytotoxicity of eukaryotic cells, low activity of antibacterial peptide, etc. Efficiently obtain and improve the effect of safety

Inactive Publication Date: 2014-09-03
科美博瑞科技(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, existing studies have shown that many natural antimicrobial peptides are not highly active or have high cytotoxicity to eukaryotic cells. Therefore, molecular improvements need to be used to further increase their activity or reduce their activity before they can be developed as new antibiotic substitutes. its toxicity
In addition, bacterial infection has become an important reason affecting animal husbandry production, especially the emergence of drug-resistant strains, which increases the difficulty of disease treatment

Method used

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  • PAMP37-PR39 fused antibacterial peptide and preparation method thereof
  • PAMP37-PR39 fused antibacterial peptide and preparation method thereof
  • PAMP37-PR39 fused antibacterial peptide and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Structural Design and Prediction of Antimicrobial Peptides

[0033] In order to enhance the bactericidal activity of the antimicrobial peptide, the porcine antimicrobial peptide (PAMP37 and PR39) were constructed in series, and the amino acid sequence of the fusion polypeptide obtained by the construction was SEQ ID1:

[0034] GLLSRLRDFLSDRGRRLGEKIERIGQKIKDLSEFFQSRRRRPPYLPRPRPPPFFPPRLPPRIPPPGFPPRFPPRFP.

[0035] Using the codon preference of Escherichia coli, the recombinant DNA sequence (SEQ ID2) of the sequence was designed, and the N-terminus of the polypeptide was predicted to be an α-helix, and the C-terminus was a linear structure.

Embodiment 2

[0036] Example 2 Construction of Antimicrobial Peptide Expression Vector

[0037] 1) Optimize the DNA sequence to adapt to the codon preference of Escherichia coli, increase the restriction site used for vector construction, and design the fusion polypeptide DNA sequence (SEQ ID3) as follows:

[0038] GGTACC GGTCTGCTGTCTCGTCTGCGTGACTTCCTGTCTGACCGTGGTCGTCGTCTGGGTGAAAAAATCGAACGTATCGGTCAGAAAATCAAAAGACCTGTCTGAATTCTTCCAGTCTCGTCGTCGTCCGCGTCCGCCGTACCTGCCGCGTCCGCGTCCGCCGCCGTTCTTCCCGCCGCGTCTGCCGCCGCGTATACCACCAAGGCTTTCCACCAAGGTTCCCACCACGATTCCCA TAACTCGAG

[0039] The sequences underlined in black are restriction sites (KpnI and XhoI). Sequences are sent to biological companies for synthesis. 3) Construction of antimicrobial peptide expression vector

[0040] The pET30a vector and the synthesized sequence were respectively digested with KpnI and XhoI, and after recovering the digested products, they were ligated to construct the pET30a-P37-PR39 expression vector. The process was as...

Embodiment 3

[0041] Example 3 Induced expression of antimicrobial peptides

[0042] 1) Transform the above expression vector into Escherichia coli BL21 according to conventional methods, spread the transformed bacteria on LB plates, and culture them overnight in an incubator at 37°C. Clearly positive clones were observed growing on the plate.

[0043] 2) Pick a positive single colony and culture it in LB liquid until the OD600 is 0.4-0.6, then add 0.5mM IPTG, induce the expression of the target protein at 250rpm overnight at 37°C, and make a control without adding IPTG.

[0044]3) Collect the bacteria, centrifuge at 10000 g for 15 minutes and discard the supernatant. Sonicate the bacteria in 1M Tris-HCl solution, centrifuge at 20,000g for 30 minutes, analyze the supernatant by SDS-PAGE, and stain the polyacrylamide gel with Coomassie Brilliant Blue to see obvious soluble expression bands of the target protein.

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Abstract

The invention relates to a PAMP37-PR39 fused antibacterial peptide and a preparation method thereof. Pig antibacterial peptides PAMP37 and PR39 are in series connected to form a fused antibacterial peptide; by utilizing preference of an escherichia coli codon, a DNA (deoxyribonucleic acid) sequence of the fused antibacterial peptide is designed, and is constructed into an expression vector, and then expressed and purified; an antibacterial activity result shows that the antibacterial activity of the obtained fused antibacterial peptide is greatly improved. The antibacterial peptide provided by the invention can improve disease resistance of pigs very well, can effectively substitute or reduce antibiotics for being used as a feed additive, so that animal product safety is improved, and therefore, the PAMP37-PR39 fused antibacterial peptide has an important practical application value.

Description

technical field [0001] The invention belongs to the technical field of gene engineering drug production in the biotechnology pharmaceutical industry, in particular to a PAMP37-PR39 fusion antibacterial peptide and a preparation method thereof. Background technique [0002] The widespread use of traditional antibiotics has made many pathogenic bacteria resistant to them, and the use of antibiotic additives has seriously damaged the microbial balance in the animal intestines and is easy to remain in animals, seriously affecting the quality of animal products and human health. Antibacterial peptides (antibacterial peptides) is a kind of polypeptide with strong antibacterial effect produced by organisms, and is an important component of biological innate immunity. It has the advantages of broad-spectrum bactericidal effect, small relative molecular weight, thermal stability, and good water solubility. More importantly, antimicrobial peptides have almost no effect on healthy euka...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/17A61P31/04A23K1/17C12R1/19A23K20/195
Inventor 石磊任建廷
Owner 科美博瑞科技(北京)有限公司
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