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Large yellow croaker hepcidin antibacterial peptide and preparation method thereof

A large yellow croaker and antimicrobial peptide technology, applied in the field of fish genetic engineering, can solve the problems of antimicrobial peptides without broad-spectrum antibacterial effect, high cost, complex structure, etc., and achieve the effect of increasing the molecular weight of the expression product, low price, and simple configuration

Active Publication Date: 2011-02-16
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex structure of the four disulfide bonds, it is difficult to synthesize large yellow croaker hepcidin antimicrobial peptides by chemical methods, the cost is expensive, and it is difficult to industrialize production
At present, recombinant hepcidin antimicrobial peptides obtained by genetic engineering often have no broad-spectrum antibacterial effect, or the antibacterial spectrum research is insufficient (4. Zhang H, Yuan QP, Zhu YP, Ma RY. Expression and preparation of recombinant hepcidin in Escherichia coli[ J] Protein.Expr.Purif., 2005, 41:409-416; 5. Srinivasulu B., Syvitski R., Seo J.K., Mattatall N.R., Knickle L.C., Douglas S.E., Expression, purification and structural characterization of recombinant hepcidin, an antimicrobial peptide identified in Japanese flounder, Paralichthys olivaceus, Protein Expr. Purif., 2008, 61: 36-44)

Method used

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  • Large yellow croaker hepcidin antibacterial peptide and preparation method thereof
  • Large yellow croaker hepcidin antibacterial peptide and preparation method thereof
  • Large yellow croaker hepcidin antibacterial peptide and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1pET-28a + Carrier handling

[0053] 1)pET-28a + Plasmid extraction;

[0054] Will contain pET-28a + The E.coli BL21(DE3)pLysS of the prokaryotic expression vector was stored in 20% glycerol and streaked on an LB plate containing 50μg / ml kanamycin and cultured at 37°C for 12h. Pick a single clone, inoculate it in 5ml of LB liquid medium supplemented with 50μg / ml kanamycin and 0.5% D-glucose, culture at 37°C, 180rpm for 8h, and extract the plasmid according to the instructions of the Dongsheng Plasmid Minimal Extraction Kit.

[0055] 2)pET-28a + Enzyme digestion and dephosphorylation treatment of the vector;

[0056] According to the following system for pET-28a + The plasmid is digested with NcoI-XhoI

[0057] NcoI-XhoI double digestion:

[0058] 10×K Buffer (buffer) 15μl

[0059] 0.1% BSA 15μl

[0060] NcoI endonuclease 7μl

[0061] XhoI endonuclease 7μl

[0062] pET-28a + Plasmid 100μl

[0063] Ultrapure water 6μl

[0064] Total volume 150μl

[0065] After incubating at 37°C f...

Embodiment 2

[0071] Example 2 Obtaining the target gene

[0072] 1) Design of primers:

[0073] According to the multiple cloning sites on the pET-28a+ vector and the large yellow croaker hepcidin cDNA sequence, an expression vector containing the leader peptide of the large yellow croaker hepcidin antimicrobial peptide was designed (see figure 2 ), PCR upstream and downstream primers are shown in Table 1, boldface indicates restriction enzyme sites, C-Pro-PC-hepc forward, where CCATGG is the Nco I restriction site, and the front GCG is the protection base of the restriction site , C-Pro-PC-hepc reverse where CTCGAG is the restriction site of Xho I, and the front CCG is the designed protective base:

[0074] Table 1. Upstream and downstream primers used to express C-Pro-PC-hepc

[0075]

[0076] 2) PCR amplification of hepcidin antimicrobial peptide gene sequence of large yellow croaker

[0077] Use the pPMD18-T positive plasmid containing the cDNA of large yellow croaker hepcidin as a template, a...

Embodiment 3

[0106] Example 3 Construction, transformation and identification of expression vector

[0107] Connect the treated pET-28a+ prokaryotic expression vector with the target fragment of the large yellow croaker hepcidin antimicrobial peptide cDNA containing the same sticky end. The reaction system is as follows:

[0108] pET-28a+ 2μl

[0109] Target fragment 1μl

[0110] Ultrapure water 1.8μl

[0111] T4 DNA Ligase 0.2μl

[0112] Ligation Solution I 5μl

[0113] Total volume 10μl

[0114] Ligation overnight at 16°C, the next day, take 5μl of the ligation reaction solution and transform it into 50μl E.coli TOP10F' competent cells, spread it on an LB agar plate containing 50μg / mL kanamycin and 0.5% D-glucose and incubate at 37°C overnight. A single colony was picked the next day, and positive clones were picked, cultured at 37°C and 180 rpm for 8 hours, and plasmids were extracted according to the instructions of the Dongsheng Plasmid Minimal Extraction Kit. Use the upstream and downstream pr...

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Abstract

The invention discloses a large yellow croaker hepcidin antibacterial peptide and a preparation method thereof, relating to fish gene engineering in the biotechnology field, and providing a large yellow croaker hepcidin antibacterial peptide and a preparation method thereof. The preparation method of the large yellow croaker hepcidin antibacterial peptide comprises the following steps: cloning a large yellow croaker hepcidin antibacterial peptide gene; constructing a recombinant vector; converting a host cell; selecting positive clone for prokaryotic expression; separating and purifying an expression product; and obtaining the large yellow croaker hepcidin antibacterial peptide. During separation and purification, the yield of protein is improved, thus solving the problem of protein precipitation when the hepcidin antibacterial peptide expresses renaturation. The solution has simple preparation and low cost. The prepared large yellow croaker hepcidin antibacterial peptide has high broad-spectrum antimicrobial activity, can be used for scientific researches, can serve as feed additive for preventing and curing diseases of sea farming fishes and has wide application prospect.

Description

Technical field [0001] The invention relates to fish genetic engineering in the field of biotechnology, in particular to large yellow croaker hepcidin antibacterial peptide and a preparation method thereof. Background technique [0002] Antimicrobial peptides (AMPs) are widely present in animals and plants, and they play an important role in the immune response of animals and plants. Hepcidin is a family of antimicrobial peptides rich in cysteine ​​residues at the C-terminal conserved site. In 2000, Krause A et al. (1. Krause A, Neitz S, Magert HJ, Schulz A, Forssmann WG, Knappe PS, Adermann K. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity[J]FEBS Lett., 2000, 480: 147-150) LEAP-1 (hereinafter referred to as hepcidin) was first discovered from human plasma. The C-terminus of Hepcidin antimicrobial peptide retains the enriched region formed by cysteine. The study of circular dichroism (CD) spectroscopy confirmed that hepcidin antimicrobial...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/12C07K14/46C07K1/22
Inventor 王克坚蔡灵蔡晶晶
Owner XIAMEN UNIV
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