Method for efficiently expressing recombinant antibacterial peptide PaDef with pichia pastoris

A Pichia pastoris and antimicrobial peptide technology, applied in the biological field, achieves good tolerance and good stability

Inactive Publication Date: 2017-06-06
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
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  • Claims
  • Application Information

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The technology of the present invention has no public report or use at home and abroad

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  • Method for efficiently expressing recombinant antibacterial peptide PaDef with pichia pastoris
  • Method for efficiently expressing recombinant antibacterial peptide PaDef with pichia pastoris
  • Method for efficiently expressing recombinant antibacterial peptide PaDef with pichia pastoris

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Embodiment 1

[0028] Construction of expression vectors—obtained antimicrobial peptides from antimicrobial peptide library (http: / / aps.unmc.edu / AP / ) and gene bank (https: / / www.ncbi.nlm.nih.gov / genbank / ) respectively Amino acid sequence (as shown in SEQ ID NO: 4, APD ID: AP 02332) and gene sequence (as shown in SEQ ID NO: 3, ACCESSIONNO.KC007441) of PaDef. After the gene sequence of PaDef is codon optimized and transformed, the gene sequence designed is as shown in SEQ ID NO: 1 (the order is EcoR I-BamH I-ATG-6×His-GATGACGATGACAAG-PaDef-stop codon-XhoI-Kpn I). Then it was handed over to Suzhou Jinweizhi Biotechnology Co., Ltd. for the whole gene synthesis, and inserted into the vector pUC19 to obtain the recombinant plasmid pUC-mPaDef. The recombinant plasmid pUC-mPaDef and pPICZαA empty vector were digested with EcoR I and Kpn I, respectively, at 37°C for 3h. The enzyme digestion system is 1 μL of 10×Buffer, 1.5 μg of pUC-mPaDef / pPICZαA, 0.8 μL of EcoRI, 0.8 μL of KpnI, and 10 μL of the t...

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Abstract

The invention provides a method for efficiently preparing a recombinant antibacterial peptide PaDef in pichia pastoris. The method comprises the following steps: taking pichia pastoris as a host cell, modifying a gene sequence of the antibacterial peptide PaDef (the antibacterial peptide PaDef is named as mPaDef), inserting the antibacterial peptide mPaDef into a pPICZalphaA vector, successfully transferring the vector into pichia pastoris GS115, performing inducible expression with methanol and performing purification and activity identification on an expression product. A final product is a high expression strain of the recombinant antibacterial peptide mPaDef, and the yield of the recombinant antibacterial peptide in supernatant of fermentation liquor is 84mg/L. The prepared recombinant antibacterial peptide has broad-spectrum antibacterial activity and good temperature and protein stability and is comparatively good in stability in weak acid, neutral and alkaline environments.

Description

technical field [0001] The invention relates to the first successful expression of the antimicrobial peptide PaDef in Pichia pastoris through the method of genetic engineering and has broad-spectrum antibacterial activity, belonging to the field of biotechnology. Background technique [0002] Antimicrobial peptides are a class of peptide active substances produced by the host's non-specific immune defense system against exogenous pathogens, and are effector molecules of innate immunity. In recent years, with the extensive use of traditional antibiotics, the emergence of drug-resistant bacteria has brought huge hidden dangers to human health. Antimicrobial peptides have broad-spectrum anti-bacteria, fungi, viruses, and parasites, and have the advantages of not being easy to produce drug-resistant strains. At present, as a substitute for antibiotics or in synergy with antibiotics, it is expected to become a new antibacterial drug. PaDef is isolated from Mexican avocado fruit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/81C12P21/02C12R1/84
CPCC07K14/415C12N15/815
Inventor 樊振川孟德梅赵静芳凌霄代红霞
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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