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Pseudosciaena crocea hepcidin gene yeast expression product as well as preparation method and application thereof

A yeast expression and antimicrobial peptide technology is applied in the field of large yellow croaker antibacterial peptide hepcidin gene yeast expression product and its preparation, which can solve the problems of inability to express product modification and high chemical synthesis cost, and achieve the effect of obvious anti-aquatic pathogen activity and low cost.

Active Publication Date: 2013-07-31
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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AI Technical Summary

Problems solved by technology

Since the prokaryotic expression system cannot perform post-translational modification on the expression product, and the cost of chemical synthesis is generally high

Method used

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  • Pseudosciaena crocea hepcidin gene yeast expression product as well as preparation method and application thereof
  • Pseudosciaena crocea hepcidin gene yeast expression product as well as preparation method and application thereof
  • Pseudosciaena crocea hepcidin gene yeast expression product as well as preparation method and application thereof

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Embodiment Construction

[0048] 1. Acquisition of the cDNA sequence of a large yellow croaker antimicrobial peptide hepcidin gene

[0049] The applicant previously discovered multiple EST sequences encoding hepcidin through EST analysis of poly(I:C)-induced large yellow croaker spleen tissue cDNA library (5. Zheng W, Liu G, Ao J, et al. Expression analysis of Immune-relevant genes in the spleen of large yellow croaker (Pseudosciaenacrocea) stimulated with poly I:C[J]. Fish Shellfish Immunol, 2006, 21(4):414-430). Two-way sequencing was performed on one of the EST clones (NCBI GenBank accession number: CX348487). Sequence analysis showed that the clone encoded a full-length cDNA of a large yellow croaker hepcidin gene. Proteins made up of amino acids.

[0050] The cDNA sequence and deduced amino acid sequence of the large yellow croaker antimicrobial peptide hepcidin gene are as follows:

[0051]

[0052]Among them, the underlined part is the predicted signal peptide of large yellow croaker antimi...

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Abstract

The invention provides a pseudosciaena crocea hepcidin gene yeast expression product as well as a preparation method and application thereof, and relates to a pseudosciaena crocea hepcidin gene. The preparation method comprises the following steps of: cloning a full-length cDNA (Complementary Deoxyribonucleic Acid) sequence of the gene, amplifying a gene segment, then cloning into a pichia pastoris expression vector, and constructing to obtain a recombinant expression vector; converting a linearized recombinant expression vector into a pichia pastorris SMD1168 strain by electric shock, screening, carrying out PCR (Polymerase Chain Reaction) amplification and identification on a genome to obtain pichia pastorris SMD 1168 / pPICZalpah A-hepcidin, converting the expression vector by electric shock into pichia pastorris SMD 1168 strain serving as a contrast strain, inducing and culturing in a culture medium, centrifuging to collect supernatant, and obtaining a secreting type expression in the supernatant; purifying a recombination protein; adding NaCl to the supernatant till the final concentration is 0.2M, filtering liquid supernatant passing through columns to be subjected to column chromatography isolation, eluting a target protein, and dialyzing the purified protein, thus obtaining the pseudosciaena crocea hepcidin gene yeast expression product.

Description

technical field [0001] The invention relates to large yellow croaker antimicrobial peptide gene, in particular to a yeast expression product of large yellow croaker antimicrobial peptide hepcidin gene and its preparation method and application. Background technique [0002] Hepcidin is a new class of small molecule antibacterial peptides with unique properties discovered in recent years. Krause et al. (1.Krause A, Neitz S, , et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity [J]. FEBS Lett, 2000, 480:147-150) isolated and purified this peptide from human blood for the first time, and named it Liver-expressed antimicrobial peptide (LEAP1). Subsequently, Park et al. (2. Park CH, Valor EV, Waring AJ, et al. Hepcidin, a urinary antimicrobial peptide synthesized in the liver [J]. J BiolChem, 2001, 276 (11): 7806-7810) will be obtained from human urine The isolated polypeptide was named hepcidin, and this name has been used ever since...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/12C12N15/81C07K14/46C07K1/22A61P31/04C12R1/84
Inventor 陈新华汪玉华敖敬群
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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