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Pesticin and phage lysozyme fusion protein and encoding gene and application thereof

A fusion protein and plaguein technology, applied in the direction of peptide/protein components, enzymes, enzymes, etc., to achieve the effect of high bacterial lysis activity

Inactive Publication Date: 2016-01-13
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, no research on this fusion protein has been reported.

Method used

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  • Pesticin and phage lysozyme fusion protein and encoding gene and application thereof
  • Pesticin and phage lysozyme fusion protein and encoding gene and application thereof
  • Pesticin and phage lysozyme fusion protein and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Construction and expression of recombinant expression plasmid pET28a-Pst-E

[0043] 1. Design and synthesis of primers

[0044] According to the sequences of the pestin gene (Pst) and the phage lysozyme gene (e), primers were designed and synthesized (Table 1). Primers F1 and F2 were used to amplify the ORF of the Pst gene, and primers F3 and F4 were used to amplify the ORF of the e gene. The sequences of the primers F1 and F4 carried EcoRI and XhoI restriction sites (underlined parts) respectively.

[0045] Table 1 The primer sequences of the pestin gene (Pst) and the bacteriophage lysozyme gene (e)

[0046]

[0047] 2. Amplification of the target gene

[0048] Using the plasmid Pst-T carrying the pestin gene and the plasmid e-T carrying the phage lysozyme gene as templates, the primers designed in step 1 were used to perform PCR reactions on F1 / F2 and F3 / F4 to amplify the pestin gene respectively Pst and phage lysozyme gene e. The reaction...

Embodiment 2

[0066] Example 2. Activity detection of fusion protein Pst-E

[0067] (1) Detection of antibacterial activity of fusion protein Pst-E

[0068] Staphylococcus aureus ATCC25923 (purchased from China Veterinary Drug Control Institute) and Escherichia coli with serotype O78 (separated and preserved by the Laboratory of Microbiology and Immunology, College of Animal Science and Technology, Qingdao Agricultural University) were cultured to the logarithmic phase (the concentration of bacteria liquid reached 108cfu / mL), take 200 μL of bacterial solution and evenly spread it on the surface of ordinary agar plate, place a sterilized Oxford cup (8 mm in diameter) on the surface of the medium, add 200 μL of 0.3 mg / mL Pst-E obtained in Example 1 Protein solution, with the plaguein protein Pst solution (expressed and preserved by the Laboratory of Microbiology and Immunity, College of Animal Science and Technology, Qingdao Agricultural University) as the protein control, and kanamycin...

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Abstract

The invention relates to a pesticin and phage lysozyme fusion protein and an encoding gene and an application thereof. The fusion protein sequentially contains a pesticin protein Pst of yersinia pestis and a lysozyme e of a phage Bp7 from an amino terminal to a carboxyl terminal; the fusion protein gene is composed of 990 nucleotides. The amino acid sequence of the fusion protein is encoded, so that the obtained fusion protein has a relatively good effect in preventing or / and treating staphylococcus aureus.

Description

technical field [0001] The present invention relates to the field of biotechnology, relates to a recombinant fusion protein and its use, in particular to a fusion protein formed by plaguein protein Pst and bacteriophage lysozyme e and a gene encoded by the fusion protein, and the fusion protein is resistant to golden yellow Application in Staphylococcus. Background technique [0002] Phage is a general term for a class of microbial viruses that can infect bacteria, fungi, actinomycetes, or spirochetes. As a substitute for antibiotics, it has potential development value. Phage mainly acts on the peptidoglycan component in the bacterial cell wall through lysozyme, causing bacterial lysis. Lysozyme is produced in the late stage of biosynthesis in the bacteriophage lysis cycle and can be highly purified. It has the advantages of short action time when killing bacteria, is not easy to inactivate, is not limited by bacterial resistance, and has no side effects on humans and anima...

Claims

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Application Information

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IPC IPC(8): C07K14/24C12N9/36C12N15/62C12N15/70C12N1/21A61K38/16A61K38/47A61P31/04
CPCA61K38/00C07K14/24C12N9/2462C12Y302/01017Y02A50/30
Inventor 张灿任慧英王婷孙虎芝卢国民孙蓉蓉刘文华于杰
Owner QINGDAO AGRI UNIV
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