Mytilus edulis G-type lysozyme gene and recombinant protein and application thereof

A technology of gene recombination and lysozyme, applied in the field of lysozyme, to achieve the effect of strong inhibitory activity

Inactive Publication Date: 2013-06-19
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

At present, there are few researches on G-type lysoz

Method used

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  • Mytilus edulis G-type lysozyme gene and recombinant protein and application thereof

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preparation example Construction

[0011] The invention also provides a preparation method and application of the blue mussel G-type lysozyme. The preparation method can obtain the recombinant blue mussel G-type lysozyme with high purity, which can be specifically applied in the development of food preservatives and feed additives.

[0012] The technical scheme adopted by the present invention to solve the above-mentioned technical problems is: G-type lysozyme from purple mussels, including extraction of total RNA from purple mussels infected with pathogenic bacteria, mRNA purification, cDNA template preparation, G-type lysozyme gene screening, gene cloning, The steps of recombinant plasmid construction, recombinant gene expression and recombinant protein purification obtain the G-type lysozyme, and the amino acid sequence of the G-type lysozyme is shown in the sequence table SEQ ID NO.2.

[0013] The preparation method of purple mussel G type lysozyme comprises the following steps:

[0014] 1) Total RNA extrac...

Embodiment

[0024] Preparation of G-type lysozyme from purple mussel:

[0025] 1. Use Trizol reagent to extract the total RNA in the purple mussel hemolymph infected with Vibrio anguillarum, and obtain the total RNA. The extraction method is carried out according to the instructions of Invitrogen's Trizol reagent: take 50 ml of the purple mussel hemolymph, centrifuge at 2000g for 10 minutes, Discard the supernatant and add 20ml of (Invitrogen), disperse the cells with a high-speed disperser, shake vigorously at room temperature for 10 seconds; add 4ml of chloroform, shake vigorously for 30 seconds; centrifuge at 10,000g at 4°C for 10 minutes at high speed; draw the supernatant into a new centrifuge tube, add An equal volume of isopropanol (about 15ml) that has been ice-bathed, placed at -20°C for more than 1 hour; 4°C, 10,000g high-speed centrifugation for 10 minutes; carefully discard the supernatant; wash the precipitate with 5ml of 70% ethanol ; Centrifuge at 10,000g at 4°C for 10 mi...

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Abstract

The invention relates to lysozyme, specifically to a Mytilus edulis G-type lysozyme gene and a recombinant protein and application thereof. The G-type lysozyme gene has a base sequence as represented by SEQ ID No. 1 in a sequence table. Through PCR technology, a gene fragment coding the mature peptide of the G-type lysozyme gene is amplified and cloned into a pET21(a) expression vector, and prokaryotic in-vitro recombinant expression in escherichia coli BL21(DE3)plysS is realized. After Ni-affinity chromatographic purification and dialysis renaturation, a recombination product has inhibitory effects on a variety of Gram positive and negative pathogens and especially has a substantial effect on pseudomonas putida, and minimal inhibitory concentration is 2.39 to 4.79 mu mol/L. The invention provides bases for further research on immune defense mechanism of economic shellfish and lays a foundation for disease control, genetic breeding and feed additives for aquatic livestock.

Description

technical field [0001] The invention relates to lysozyme, in particular to a blue mussel G-type lysozyme gene and its recombinant protein and application. Background technique [0002] Lysozyme (Lysozyme, EC 3.2.1.17) is one of the very important non-specific immune factors in invertebrates. It mainly cuts off the β- The connection between 1,4 glycosidic bonds destroys the peptidoglycan scaffold, causing cell swelling and causing bacterial lysis. Lysozyme can not only directly kill bacteria during the body's immune response, but also induce and regulate the synthesis and secretion of other immune factors. Lysozyme is divided into six categories according to its source: C (chicken) type, G (goose) type, I (invertebrate) type, plant type, bacterial type and T4 phage type lysozyme. Lysozymes in the animal kingdom include the first three types. Type C lysozymes mostly come from vertebrates and arthropods; type G lysozymes mainly exist in mammals, birds and fish; and type I ly...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/36A61K38/47A61P31/04A23L3/3571A23K1/165
Inventor 赵建民吴惠丰王清张林宝
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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