High efficient preparation of novel antimicrobial peptide Mytichitin-A in pichia pastoris

A technology of Pichia pastoris and antimicrobial peptides, which can be applied in the direction of antibacterial drugs, medical preparations containing active ingredients, peptides, etc., can solve the problems of undisclosed use and achieve high-efficiency expression and broad market prospects

Inactive Publication Date: 2017-08-01
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • High efficient preparation of novel antimicrobial peptide Mytichitin-A in pichia pastoris
  • High efficient preparation of novel antimicrobial peptide Mytichitin-A in pichia pastoris
  • High efficient preparation of novel antimicrobial peptide Mytichitin-A in pichia pastoris

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Embodiment 1

[0024] Construction of expression vector - add 6×His tag, enterokinase gene sequence, EcoRI restriction site and start codon to the 5' end of the target gene Mytichitin-A; introduce stop codon and KpnI restriction site at 3' point, according to SEQ ID NO: 2, the whole gene synthesis was carried out, and inserted into the vector pUC19 to obtain the recombinant plasmid pUC-Mytichitin-A. The recombinant pUC-Mytichitin-A and pPICZαA empty vectors were subjected to double enzyme digestion (EcoRI, KpnI) respectively, at 37°C for 3h. Enzyme digestion system is 10×Buffer 1μL, pUC-Mytichitin-A / pPICZαA 1.5μg, EcoRI 0.8μL, KpnI 0.8μL, total system 10μL; recover the ligation, target gene Mytichitin-A fragment and vector pPICZαA fragment are ligated for 2h at room temperature or 4℃ Overnight ligation, ligation system: pPICZαA 20ng, Mytichitin-A 8ng, T4 ligase 0.8μL, total system 10μL. The pPICZα-Mytichitin-A recombinant plasmid was successfully constructed. The recombinant plasmid pPICZα...

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Abstract

The invention discloses a method for high efficient preparation of novel Mytilus coruscus antimicrobial peptide Mytichitin-A in pichia pastoris. A gene engineering method is adopted and the full-length gene of the antimicrobial peptide Mytichitin-A is inserted into an exosome type expression vector pPICZalphaA through EcoRI and KpnI restriction enzyme cutting sites, so as to obtain a pPICZalphaA-Mytichitin-A recombinant expression vector. Through an electrotransformation method, a SacI enzyme linearized recombinant plasmid pPICZalphaA-Mytichitin-A is transferred into the pichia pastoris GS115. Through optimizing, the optimum expression conditions are determined as follows: methanol concentration is 1.0%, fermentation time is 96h, and the yield is 45.5 microgram/mL. The result of bacteriostasis experiments shows that the recombinant antibacterial peptide has an obvious inhibition effect on both staphylococcus aureus and bacillus subtilis 151-1.

Description

technical field [0001] The invention relates to the antimicrobial peptide Mytichitin-A which is successfully expressed in Pichia pastoris through a genetic engineering method for the first time and has strong antibacterial activity, belonging to the field of biotechnology. Background technique [0002] Antimicrobial peptides are active polypeptides produced by body cells to fight exogenous pathogenic bacteria under inducing conditions. In recent years, there have been more and more studies on antimicrobial peptides, not only because of their functions in killing bacteria and fungi, anti-virus, anti-tumor, etc., but also because they are easily digestible in the human body, have no toxic side effects, and are heat It has many advantages such as good stability, no drug residue, and is not easy to produce drug resistance, making it recognized as the most potential substitute for antibiotics and food preservatives in the world, with huge development potential. Mytichitin-A is i...

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Application Information

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IPC IPC(8): C07K14/435C12N15/81C12N1/19A61K38/17A61P31/04A23L3/3526C12R1/84
CPCC07K14/43509A23L3/3526A61K38/00C12N15/815
Inventor 樊振川孟德梅代红霞高晓芳杨永海
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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