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Scylla serrata antibacterial peptide and its genes and clone method for genes

A technology of Scylla serrata and cloning method, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., and can solve the problems of difficult to obtain antimicrobial peptides and high cost

Inactive Publication Date: 2007-07-11
XIAMEN UNIV
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Problems solved by technology

Therefore, antimicrobial peptides will be an antibacterial drug with broad application prospects in the prevention and treatment of marine crab diseases; however, natural antimicrobial peptides are extracted directly from crabs or artificially synthesized by chemical methods, which are limited by animal sources and chemical synthesis methods , it is difficult to obtain a sufficient amount of antimicrobial peptides, and the cost is expensive. Therefore, isolating and cloning antimicrobial peptide genes and using genetic engineering methods to produce large quantities of antimicrobial peptides in vitro is the key to obtaining high-yield antimicrobial peptides and ensuring their widespread application in aquaculture. premise

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  • Scylla serrata antibacterial peptide and its genes and clone method for genes

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Embodiment

[0081] 1. Separation and purification of scygonadin antimicrobial peptide from Scylla serrata:

[0082] Dissect and separate the vas deferens of Scylla serrata, add an equal volume of acidic extract [15% acetic acid, 0.1% trifluoroacetic acid and 1 mM benzoic acid sulfonyl fluoride (PMSF)], and homogenize for 20 min (6000r·min -1 ), the homogenate was centrifuged at 15,000 g for 45 min at 4°C, and the supernatant was taken. The precipitate was resuspended with an equal volume of extract, ultrasonically crushed (15 times at 400w 10s), centrifuged at 15000g at 4°C for 45min, the supernatant was collected, and the two supernatants were combined and freeze-dried. Dissolve the sample with 0.05M sodium acetate buffer (pH5.0), centrifuge at 10000g at 4°C for 10min, take the supernatant, pass through the SP-Sepharose Fast Flow ion-exchange chromatography column, and elute with 1.5M ammonium acetate gradient, 0-50% Elution was 5 column volumes (CV), 50%-80% elution 2CV, 80-100% elutio...

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Abstract

The present invention Scylla serrata antibiotic peptide and its gene and genetic cloning method, and relates to one kind of active antibiotic peptide scygonadin and its gene. Antibiotic peptide scygonadin is separated and purified from male sexual gland of Scylla serrata, and degenerate primer is designed and synthesized based on the total RNA of the male sexual gland as template and the detected 20 amino acid sequence in N end of antibiotic peptide scygonadin and cloned to the whole length cDNA gene sequence one the antibiotic peptide scygonadin by means of RT-PCR, RACE or other molecular biological process. Genetic engineering strain with high efficiency expression of the antibiotic peptide may be constituted via constituting eukaryotic expression vector of the gene so as to externally produce antibiotic peptide as feed additive for aquatic animal to resist drug resisting bacteria. In addition, the present invention may be used also in transgenic blue crab breeding.

Description

technical field [0001] The invention relates to an antibacterial activity polypeptide scygonadin isolated and purified from the male gland of Scylla serrata (forskal), and a gene encoding the antibacterial peptide cloned accordingly. Background technique [0002] Scylla serrata, English name mud crab, blue crab Latin name Scylla serrata (forskal), commonly known as blue crab, hairy crab, chrysalis, and scorpion, has a wide natural distribution and can be found in temperate, subtropical, and tropical sea areas. It is an important marine aquaculture economic species. In recent years, with the breakthrough of blue crab artificial breeding technology and the improvement of breeding technology, blue crab farming has gradually changed from extensive farming to intensive farming. However, with the increase of breeding density, the phenomenon of eutrophication in the breeding water body is becoming more and more serious, which leads to the deterioration of the breeding environment,...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12P19/34A61P31/00
Inventor 王克坚黄文树李少菁王桂忠
Owner XIAMEN UNIV
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