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Pichia pastoris-optimized oncorhynchus mykiss hepcidin, oncorhynchus mykiss hepcidin-containing expression carrier, oncorhynchus mykiss hepcidin-containing strain and applications

A Pichia pastoris and expression vector technology, applied in the field of genetic engineering, can solve the problem that Escherichia coli has no obvious bacteriostatic effect and the like

Inactive Publication Date: 2014-03-26
BEIJING FISHERIES RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0012] In 2007, Zhang Hui et al. designed and synthesized the human hepcidin gene sequence, and constructed the secreted recombinant Pichia pastoris expression vector pPICZαA-hep. The expression level reached 100 mg / L after induction in the Pichia pastoris host strain GS115. It has antibacterial activity against Bacillus subtilis, but has no obvious antibacterial effect against Escherichia coli (Zhang Hui et al., Acta Biological Engineering, 2007, 23: 381-385)

Method used

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  • Pichia pastoris-optimized oncorhynchus mykiss hepcidin, oncorhynchus mykiss hepcidin-containing expression carrier, oncorhynchus mykiss hepcidin-containing strain and applications
  • Pichia pastoris-optimized oncorhynchus mykiss hepcidin, oncorhynchus mykiss hepcidin-containing expression carrier, oncorhynchus mykiss hepcidin-containing strain and applications
  • Pichia pastoris-optimized oncorhynchus mykiss hepcidin, oncorhynchus mykiss hepcidin-containing expression carrier, oncorhynchus mykiss hepcidin-containing strain and applications

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Experimental program
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Effect test

Embodiment 1

[0063] Example 1. Optimization and cloning of rainbow trout antimicrobial peptide hepcidin gene

[0064] Rare codon optimization for target genes

[0065] Using the codon preference type of Pichia pastoris, use the online software http: / / gcua.schoedl.de / to complete the sequence of the rainbow trout antimicrobial peptide hepcidin gene (GenBank accession no.HQ711993, figure 1 ) for rare codon analysis, the results are as follows figure 2 (A) shown. The figure also compares the codon preference of Pichia pastoris and exogenous genes, in which the red is the exogenous gene, and the black is the Pichia pastoris. It is acceptable for the black bars to be 0.5 to 2.0 times the value of the red bars. According to the degeneracy of codons, the bases of amino acids that are not suitable for expression are optimized and adjusted. The diversity of the target gene hepcidin in Pichia pastoris was optimized from the initial 46.88% to 35.23%. The optimization results are as follows fi...

Embodiment 2

[0078] Embodiment 2, construction of recombinant expression vector

[0079] Construction of recombinant expression vector pGAPZαA-hep / mhep

[0080] use SV Gel and PCR Clean-Up Kit, respectively purify the PCR products of the amplified hep / mhep target gene. Then, the purified product and plasmid pGAPZαA DNA were double-digested with XhoI enzyme and XbaI enzyme, respectively, and then the two DNA fragments were ligated at 16°C for 2 h using T4 ligase to obtain recombinant plasmids pGAPZαA-hep and pGAPZαA-mhep. build process reference Figure 5 shown.

[0081] Double enzyme digestion reaction system

[0082]

[0083] T4 Ligase Reaction System

[0084]

[0085] The competent Escherichia coli cells Trans5α purchased from Beijing Quanshijin Biotechnology Co., Ltd. were transformed into Escherichia coli by heat stimulation. Sequence the gene of interest on the recombinant plasmid using universal sequencing primers. Using the software DNAman to compare the sequencing result...

Embodiment 3

[0088] Embodiment 3, transformation, expression and screening of recombinant Pichia pastoris

[0089] Electrotransformation of recombinant Pichia pastoris

[0090] The constructed recombinant plasmids were extracted, and pGAPZαA-hep / mhep was linearized by digestion with BspH I, and pPICZαA-hep / mhep was linearized by digestion with Sac I. The single enzyme digestion reaction system is as follows, and the reaction condition is to incubate at 37° C. for 2-3 hours.

[0091] BspH I and Sac I single enzyme digestion reaction system

[0092] reaction system Volume (μL) 10×NEB Buffer4 5.0 BspH I / Sac I 1.0 Recombinant plasmid pGAPZαA / pPICZαA-hep / mhep 20.0 wxya 2 0 24.0 total capacity 50.0

[0093] According to the instructions of pPICZαA, Pichia pastoris competent cells were prepared, and the recombinant plasmids pGAPZαA-hep / mhep and pPICZαA-hep / mhep were electrotransformed into Pichia pastoris GS115 (Mut + type, methanol utilization fa...

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Abstract

The invention provides a pichia pastoris-optimized oncorhynchus mykiss hepcidin gene sequence. Four recombinant plasmids pGAPZAlpha A / pPICZAlphaA-hep and pGAPZAlphaA / pPICZAlphaA-mhep are constructed by utilizing hepcidin genes or segments thereof, and are respectively electrotransformed into pichia pastoris GS115 and KM71H host cells to obtain a recombinant pichia pastoris strain, the recombinant pichia pastoris strain is subjected to culture expression to obtain expression polypeptide with the multi-bacteria resistance activity. The expression polypeptide has stronger bacteriostatic activity on escherichia coli, staphylococcus aureus and a part of fish pathogenic bacteria (vibrio parahaemolyticus 1.2164, aeromonas veronii X-1-06909, aeromonas hydrophila HA-336, and aeromonas veronii CL0901).

Description

technical field [0001] The invention belongs to the field of genetic engineering. Specifically, the invention relates to a gene sequence of rainbow trout antimicrobial peptide hepcidin optimized by Pichia pastoris, an expression vector containing it, a transformant containing it and a polypeptide expressed thereby. Background technique [0002] Rainbow trout (Oncorhynchus mykiss) is a cold-water fish of the family Salmonidae, known as "ginseng in water". It has delicious meat, rich nutrition, high protein and fat content, and almost zero cholesterol content. It is one of the four excellent freshwater cultured species promoted by the Food and Agriculture Organization of the United Nations to the world. [0003] With the development of my country's aquaculture industry, high-density and intensive aquaculture has caused a series of problems, such as the pollution of the water environment is becoming more and more serious, leading to the deterioration of the living environment o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/81C12N1/19A61K48/00A61K38/17A61P31/04C12R1/84
Inventor 罗琳姜娜马志宏李铁梁邢薇
Owner BEIJING FISHERIES RES INST
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