Eukaryotic fused expression product of two marine animal antibacterial peptide genes, and preparation method thereof

A marine animal, fusion expression technology, applied in eukaryotic fusion expression products and preparation, in the field of antibacterial peptides of Scylla pseudocaveus, can solve the problems of affecting the antibacterial activity of Hepcidin, affecting the antibacterial function of the combined polypeptide Scy-hepc, and achieve potential application value Effect

Active Publication Date: 2012-01-04
XIAMEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The recombinantly expressed Hepcidin in the prokaryotic expression system of Escherichia coli may affect the antibacterial activity of Hepcidin ...

Method used

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  • Eukaryotic fused expression product of two marine animal antibacterial peptide genes, and preparation method thereof
  • Eukaryotic fused expression product of two marine animal antibacterial peptide genes, and preparation method thereof
  • Eukaryotic fused expression product of two marine animal antibacterial peptide genes, and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Construction of Pichia pastoris recombinant expression plasmid pPIC9K-scy / hepc

[0068] 1) Acquisition of the scygonadin gene in the mud crab and the hepcidin gene in the large yellow croaker

[0069] According to the multiple cloning site on the pPIC9K vector, according to the cDNA sequence of Scygonadin (Genbank accession number: AY864802 ) and the cDNA sequence of large yellow croaker hepcidin (Genbank accession number: EF156401 ) Design the specific upstream and downstream primers of scygonadin and hepcdin combined polypeptide. The connecting peptide (linker) of the two tandem expression genes is 6 amino acid sequences: GGPGSG, and the corresponding codon sequence is (according to the codon preference of Pichia pastoris [4，5] Codon optimized): 5'GGTGGCCCAGGTTCCGGT3'.

[0070] F1: 5′GGG GGCCAGGCACTCAACAA3', black italics indicate the introduced EcoR I restriction site.

[0071] A connecting peptide gene sequence containing 6 amino acids was added to...

Embodiment 2

[0099] Example 2 Induced expression of pPIC9K-scy / hepc recombinant plasmid in Pichia pastoris GS115

[0100] 1) Linearization of pPIC9K-scy / hepc

[0101] The strains containing the expression vector with correct sequencing were streaked and cultured, and the single clones were picked and shaken for culture. After the plasmid was extracted, 10-20 μg of the plasmid was linearized by the Sac I restriction endonuclease. The reaction system was as follows:

[0102]

[0103] The linearized pPIC9K-scy / hepc was recovered with a nucleic acid co-precipitation agent. Then it was transformed into Pichia pastoris GS115 competent cells by electric shock method, and its expression was induced.

[0104] 2) Induced expression of combined polypeptide Scy-hepc

[0105] (1) Induced expression of Scy-hepc (changes in expression at different induction times):

[0106] ①Select several positive clones grown on MD plates and inoculate them in 6ml of BMGY (pH6.0) medium, culture at 29°C and 230rp...

Embodiment 3

[0114] Example 3 Purification of target protein Scy-hepc by affinity chromatography

[0115] 1) Preparation of samples before column loading

[0116] After pPIC9K-scy / hepc recombinant plasmid transformed Pichia pastoris GS115, under the condition of pH 6.0, 0.75% methanol induced expression for 36 hours, centrifuged to remove the precipitate, and collected the fermentation supernatant. At 4°C, dialyze the Pichia pastoris induced expression supernatant with PBS dialysate (50mM phosphate buffer + 50mMNaCl, pH9.0) for 2 to 3 times, centrifuge, collect the supernatant, filter through a 0.45μm membrane filter, and put it on the column for purification .

[0117] 2) Purify the target protein Scy-hepc by affinity chromatography

[0118] Affinity purification with nickel ion chelate affinity chromatography column, chromatographic reagents are:

[0119] Solution A: 20mM phosphate buffer + 50mM NaCl + 10mM imidazole, pH8.5;

[0120] Solution B: 20mM phosphate buffer + 500mM NaCl + 1...

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Abstract

The invention discloses a eukaryotic fused expression product of two marine animal antibacterial peptide genes, and a preparation method thereof, and relates to genetic engineering expressions of antibacterial peptides of marine fishes and crabs. The two marine animal antibacterial peptide genes comprise mud crab antibacterial peptide scygonadin gene, a connecting peptide, large yellow croaker antibacterial peptide hepcidin gene. The preparation method comprises the following steps that: the mud crab antibacterial peptide scygonadin gene and the large yellow croaker antibacterial peptide hepcidin gene are amplified through PCR reactions; a overlap PCR reaction is adopted for preparing the target gene fragment of a combined polypeptide Scy-hepc; the resulting fusion gene Scy-hepc is inserted into an expression vector to construct a fusion gene Scy-hepc-carried recombinant expression vector; the resulting recombinant expression vector is transferred into the host cell, and then the induction expression is adopted for the host cell to obtain the expression product; the expression product is isolated and purified to obtain the recombinant protein, which is the combined polypeptide Scy-hepc.

Description

technical field [0001] The present invention relates to the genetic engineering expression of marine fish and crab antimicrobial peptides, in particular to the eukaryotic fusion expression product and preparation method of the antimicrobial peptide Scygonadin from Scylla pseudocaveus and the antimicrobial peptide Hepcidin from large yellow croaker in Pichia pastoris GS115 with antibacterial activity . Background technique [0002] Antimicrobial peptides (antimicrobial peptides) are important components of innate immunity, widely exist in various categories of biology, relatively conservative in evolution, and have broad-spectrum antibacterial, antiviral and antitumor effects. The research, development and application of antimicrobial peptides have important scientific significance and practical application value for solving the problems of bacterial drug resistance in mariculture production, drug residues in aquatic products and water environment pollution caused by the abus...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/12C12N15/81C07K19/00C07K1/22
Inventor 王克坚彭会陈贝
Owner XIAMEN UNIV
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