Recombination expression of cynoglossus semilaevis antibacterial peptide hepcidin and application thereof

A technique of semi-smooth tongue sole and antimicrobial peptide, which is applied in the field of recombinant expression of the semi-smooth tongue sole antimicrobial peptide hepcidin, can solve the problems of high cost, complicated synthetic process of antimicrobial peptide, difficult mass production and the like, and achieves the effect of solving the harm of diseases

Inactive Publication Date: 2013-04-10
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a recombinant expression and application of the antibacterial peptide hepcidin of semi-smooth tongue sole, aiming at the deficiencies in the prior art that the antimicrobial peptide synthesis process is complicated, the cost is high, and it is not easy to produce on a large scale, and the semi-smooth tongue sole antibacterial peptide hepcidin mature peptide obtained by cloning is used The gene sequence is used to construct prokaryotic expression vectors, so as to obtain a large number of active antimicrobial peptides, which can be used in aquatic organisms or poultry animal breeding, or used as a substitute for antibiotics for biological disease control, bacterial and even viral diseases. prevention

Method used

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  • Recombination expression of cynoglossus semilaevis antibacterial peptide hepcidin and application thereof
  • Recombination expression of cynoglossus semilaevis antibacterial peptide hepcidin and application thereof

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Experimental program
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Effect test

Embodiment 1

[0018] Construction of embodiment 1 recombinant expression vector, identification and expression of antimicrobial peptide, purification

[0019] 1. Construction and identification of the recombinant plasmid: According to the mature peptide cDNA sequence (SEQ ID NO: 2) of the antibacterial peptide hepcidin of half-smooth tongue sole, it was modified according to the codon preference of Escherichia coli, and a pair of specific primers P1 and P2 were used to introduce The restriction endonuclease sites EcoRI and NotI sites are used to amplify the mature peptide of the antimicrobial peptide of half-smooth tongue sole, and the primer sequence is:

[0020] P1: 5′-GGAATTCCGTGTGAAGCGC-3′

[0021] P2: 5'-TATGCGGCCGCTTAGTATTTACAAC-3';

[0022] The PCR reaction volume was 25ul, and the amplification conditions were: 94°C for 5min; 94°C for 30s, 55°C for 30s, and 72°C for a total of 28 cycles; 72°C for 7min. After the PCR product was detected by agarose electrophoresis, the product was ...

Embodiment 2

[0029] Antibacterial activity assay of embodiment 2 recombinant protein

[0030] Use the agar hole diffusion method to detect the antibacterial activity of the antibacterial peptide hepcidin recombinant protein of half-smooth tongue sole: 200uL of the suspension of the test bacteria in the logarithmic growth phase (OD600=0.2~0.3) was mixed with LB solid medium (or seawater) at 55°C Base 2216E) 25mL was mixed evenly and plated. After it was solidified, a hole was punched with an Oxford cup (diameter 8mm), and about 1.5uM of purified recombinant antimicrobial peptide was added dropwise to the hole, and incubated at 37°C (or 28°C) for 8-12h. The same volume of PBS was used as a negative control, and the diameter of the inhibition zone was measured on the second day.

[0031] The tested bacteria include common bacteria: Escherichia coli GM109 (E.coli), Staphylococcus aureu (Staphylococcus aureu), and major marine pathogens: Vibrio harveyi (Vibrio harveyi), Vibrio parahaemolyticus ...

Embodiment 3

[0032] Example 3: Application of recombinantly produced antimicrobial peptides in the preparation of antibacterial products

[0033] Escherichia coli containing the fusion protein expression vector can be produced as a powder by methods such as microfiltration, ultrafiltration, spray drying, and lyophilization after conventional culture, induced expression, and purification methods provided by the present invention to obtain semi-finished products of antimicrobial peptides, or The liquid preparation is obtained after being refined and purified by biochemical methods. The obtained antimicrobial peptide semi-finished products or veterinary drug-grade finished products can be directly used as antibacterial drugs in conventional dosages for the prevention and treatment of aquatic organisms or other farmed poultry diseases.

[0034] Select fish meal, bone meal, corn meal, bran, bran, bean cake, etc. as the basic feed for aquatic organisms or farmed livestock in a conventional manne...

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Abstract

The invention relates to recombination expression of cynoglossus semilaevis antibacterial peptide hepcidin and application thereof. A nucleic acid fragment used for recombining and expressing the cynoglossus semilaevis antibacterial peptide hepcidin with the sequence of SEQ ID NO: 1 is inserted into an expression vector, recombinant protein of the cynoglossus semilaevis antibacterial peptide hepcidin is expressed in host bacteria, a simple and effective method for purifying the recombinant protein is found out, and the protein expressed by the recombinant protein is applied to antibacterial products. In the invention, the vector and the host bacteria capable of carrying out antibacterial peptide recombination expression are constructed and sifted, the fusion expression and the simple and effective purification of the cynoglossus semilaevis antibacterial peptide in prokaryote are realized; compared with the traditional polypeptide synthesis method and the method for extracting polypeptide from natural resources, the preparation method provided by the invention makes large scale production in low cost possible and can be applied to feedstuff additives of marine cultivation fish, cultivation livestock and control of bacterial diseases.

Description

technical field [0001] The invention belongs to the technical field of antibacterial recombinant proteins, and in particular relates to the recombinant expression and application of an antibacterial peptide hepcidin from tongue sole. Background technique [0002] Mariculture species live in a water environment rich in various microorganisms. Long-term survival adaptation enables them to form an effective defense function, and antimicrobial peptides are important components of the non-specific immune defense system of fish, shrimp, shellfish and other animals One of them plays an important role in defending against the invasion of marine bacteria and viruses. Antimicrobial peptides are a class of natural small peptides with strong broad-spectrum antibacterial properties. Since the Swedish scientist Boman et al. isolated the first antimicrobial peptide Cecropin from silkworm chrysalis in 1975, people have been using it again. At least 900 antimicrobial peptides have been foun...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/70A23K1/16A61K38/17A61P31/04A61P31/12C12R1/19A23K20/147
Inventor 王亚楠张全启刘旭东王旭波齐洁王志刚于海洋王兴莲
Owner OCEAN UNIV OF CHINA
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