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Large yellow croaker antimicrobial peptide hepcidin gene promoter sequence and application thereof

A gene promoter region, large yellow croaker technology, applied in the direction of using vectors to introduce foreign genetic material, DNA preparation, DNA / RNA fragments, etc., can solve the problems of unclear regulation mechanism of hepcidin gene expression, and achieve strong activity and good stability Effect

Inactive Publication Date: 2013-08-07
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression regulation mechanism of fish hepcidin gene is still unclear

Method used

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  • Large yellow croaker antimicrobial peptide hepcidin gene promoter sequence and application thereof
  • Large yellow croaker antimicrobial peptide hepcidin gene promoter sequence and application thereof
  • Large yellow croaker antimicrobial peptide hepcidin gene promoter sequence and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0024] 1. Cloning of the hepcidin gene promoter of large yellow croaker

[0025]1.1 The GenomeWalker Universal Kit kit from Clontech Company was used to construct the large yellow croaker genome single-digestion library with 4 kinds of restriction endonucleases, and the 4 kinds of DNA endonucleases used were Dra I, EcoR V, PvμII and StμI. The specific operation is:

[0026] 1.1.1 Genome purity analysis.

[0027] A: Take 1 μL of large yellow croaker genomic DNA and electrophoresis on 0.6% agarose gel.

[0028] B: Trial cutting with endonuclease Dra I. The system is:

[0029]

[0030]

[0031] 1.1.2 Enzyme digestion of genome.

[0032] A: Label five 1.5mL centrifuge tubes DL1, DL2, DL3, DL4 and a positive control.

[0033] B: The components of each endonuclease digestion system include:

[0034]

[0035] C: Vortex at low speed for 5-10s and centrifuge briefly, digest overnight at 37°C.

[0036] 1.1.3 Genomic DNA purification.

[0037] A: Add an equal volume of p...

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Abstract

The invention discloses a large yellow croaker antimicrobial peptide hepcidin gene promoter sequence and application of the large yellow croaker antimicrobial peptide hepcidin gene promoter sequence, and relates to an antimicrobial peptide hepcidin gene promoter. An cloning and activity analyzing method comprises the following steps of: building a large yellow croaker gene group mono-enzyme digestion library of four restriction endonucleases; amplifying the large yellow croaker antimicrobial peptide hepcidin gene promoter sequence from the gene group mono-enzyme digestion library and connecting an amplified polymerase chain reaction (PCR) product to a pMD18T-Simple carrier; inserting gene promoter region segments into a green fluorescent protein reporter gene carrier pEGFP-1 to ensure that enhanced green fluorescent protein genes carried on the carrier are controlled by a promoter region, wherein a constructed recombinant carrier is named pEGFP-Phepcidin; and quantitatively analyzing the activity of the promoter and lipopolysaccharide LPS and influence of virus double-chain ribonucleic acid (RNA) analogue polyI:C stimulation on the activity of the promoter by using a double-luciferase reporter gene detection system.

Description

technical field [0001] The invention relates to an antimicrobial peptide gene promoter, in particular to a large yellow croaker antimicrobial peptide hepcidin gene promoter sequence and application thereof. 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, HJ, et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity [J]. FEBS Lett, 2000, 480:147-150) isolated and purified a small molecule antimicrobial peptide from human blood for the first time, And named it liver-expressed antimicrobial peptide (LEAP1). Subsequently, Park et al. (2. Park C H, Valor e E V, Waring A J, et al. Hepcidin, a urinary antimicrobial peptide synthesized in the liver [J]. J Biol Chem, 2001, 276 (11): 7806-7810) from This small molecule antimicrobial peptide was also isolated from human urine and named hepcidin. At present, the ge...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12Q1/68C12N15/85A01K67/027
Inventor 陈新华汪玉华敖敬群
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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