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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

Active Publication Date: 2013-06-19
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few researches on G-type lysozyme and its antibacterial activity at home and abroad

Method used

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

Examples

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Effect test

preparation example Construction

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

[0014] 1) Total RNA extraction: use Trizol reagent to extract total RNA from the hemolymph of mussels infected with Vibrio anguillarum, and store it at -80°C for later use; the extraction method is carried out according to the instructions of Invitrogen's Trizol reagent;

[0015] 2) mRNA purification: Oligotex mRNA purification kit was used to purify the above-mentioned total RNA to obtain mRNA; the purification method was carried out according to the instructions of Oligotex mRNA purification kit of QIAGENE Company;

[0016] 3) Preparation of cDNA template: Reverse transcribe the above mRNA with a cDNA Synthesis Kit (purchased from Stratagene) and digest it into digested fragments. I linker ligation, EcoR I terminal phosphorylation, Xho I endonuclease digestion, etc., use the QIAEX II Agarose Gel Extraction Kit purification kit (purchased from QIAGEN) to recover the enzyme-digested...

Embodiment

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

[0025] 1. Use Trizol reagent to extract total RNA from the purple mussel hemolymph infected with Vibrio anguillarum, and obtain the total RNA. The extraction method is carried out according to the instructions of the Trizol reagent from Invitrogen Company: take 50 ml of the purple mussel hemolymph, centrifuge at 2000 g 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 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 ice The same volume of isopropanol (about 15ml) that has been bathed is placed at -20°C for more than 1 hour; 10,000g high-speed centrifugation at 4°C for 10 minutes; carefully discard the supernatant; wash the precipitate with 5ml of 70% ethanol; 4 Centrifuge at high spe...

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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 is represented by a base sequence of SEQ ID No. 1 in a sequence table. A recombination expression product of the G-type lysozyme gene can be used for preparing an antibacterial agent, an aquatic product antistaling agent or an aquatic product feed additive. Mytilus edulis G-type lysozyme in the invention can be prepared through the following steps: extraction of total RNA from Mytilus edulis infected with pathogens, purification of mRNA, preparation of a cDNA template solution, screening of the G-type lysozyme gene, gene cloning, construction of recombinant plasmid, expression of a recombinant gene and purification of the recombinant protein, and an amino acid sequence of the G-type lysozyme is represented by SEQ ID No. 2 in the sequence table. The G-type lysozyme belongs to a novel Mytilus edulis lysozyme and has powerful inhibitory activity on a variety of marine-derived Gram positive and negative pathogenic microorganisms.

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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/36A61K38/47A61P31/04A23K1/165A23L3/3571
Inventor 王清吴惠丰赵建民张林宝
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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