Application of c-type lectin-3 (cgclec-3) recombinant protein of long oyster

A technology of recombinant protein and oyster growth, applied in the field of molecular biology, can solve the problems of loss, large-scale death economy of cultured oysters, etc.

Inactive Publication Date: 2018-05-15
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, frequent outbreaks of mass mortalities in farmed oysters have caused huge economic losses

Method used

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  • Application of c-type lectin-3 (cgclec-3) recombinant protein of long oyster
  • Application of c-type lectin-3 (cgclec-3) recombinant protein of long oyster
  • Application of c-type lectin-3 (cgclec-3) recombinant protein of long oyster

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] In Vitro Prokaryotic Recombinant Expression of C-type Lectin-3 (CgCLec-3) Gene Coding Region in Oyster oyster

[0019] 1. Construction of recombinant vector

[0020] The present invention uses the pET-32a prokaryotic expression vector as the recombinant vector template. By PCR technology, gene-specific primers with specific restriction sites of BamHI and HindIII were added at the 5' ends respectively:

[0021] P1 (5'-CGCGGATCCATGGGTGCCAACTCCATTG-3'):

[0022] P2(5'-CCCAAGCTT TTATTGCGTGTTCCTCGC-3'),

[0023] The reaction conditions were as follows: 94°C pre-denaturation for 5 minutes, followed by the following cycles: 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 45 seconds, a total of 35 cycles, and finally 72°C extension for 10 minutes. The amplified fragment was purified and recovered, and connected to the pMD19-T vector. After transformation, positive clones were screened, and plasmids were extracted; plasmids were digested w...

Embodiment 2

[0037] Detection of Bacteriostatic Activity of CgCLec-3 Recombinant Protein from Oyster oyster

[0038] Escherichia coli and Staphylococcus aureus were cultured in LB medium at 37°C and 220rpm until logarithmic growth phase, and the obtained bacteria were collected by centrifugation and washed with TBS (50mM Tris-Hcl, 100mM NaCl, pH=8.0) and weighed. Suspended to a concentration of 10 4 CFU.

[0039] 50 μL of the above-mentioned or obtained long oyster recombinant protein CgCLec-3 (2 mg mL -1 or 4mg mL -1 ) with an equal volume of Escherichia coli or Staphylococcus aureus suspension in CaCl 2 Incubate at room temperature for 2 h at a final concentration of 10 mM. Take 20 μL of the above mixture in a flat-bottomed 96-well plate (Costar, Fisher), add 200 μL LB liquid medium to each well, shake and culture in a microplate reader at 37°C, measure and record the OD600 value of each well every 1 h. Another rTrx protein control group and TBS blank control group were set up. Thr...

Embodiment 3

[0041] Determination of the destructive effect of recombinant protein CgCLec-3 on Staphylococcus aureus cells

[0042] After the Staphylococcus aureus cultured to the logarithmic growth phase in the above-mentioned embodiment 2 was collected by centrifugation, it was washed and resuspended with TBS (10 4 CFU). At the same time, 50 μL of the long oyster recombinant protein CgCLec-3 (4mg mL) obtained above -1 ) with an equal volume of Staphylococcus aureus suspension in CaCl 2 Incubate at room temperature for 2 h at a final concentration of 10 mM. Set up TBS blank control and rTrx negative control. The incubated bacteria were thoroughly washed and collected by centrifugation. After fixing with 2.5% glutaraldehyde at 4°C for 24h, the bacteria were thoroughly washed with TBS, resuspended in TBS, and dropped on 24×24mm coverslips. After fixing in 2.5% glutaraldehyde for 1 h, the coverslips were rinsed in TBS for 10 min and then washed with amyl acetate for 30 min. After gradi...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to an application of C type lectin-3(CgCLec-3) recombinant protein of crassostrea gigas. The C type lectin-3(CgCLec-3) recombinant protein of the crassostrea gigas is used for preparing a bacteriostatic agent. According to the invention, the C type lectin-3(CgCLec-3) protein of the crassostrea gigas is obtained with an in-vitro recombination expression technology, and the C type lectin -3(CgCLec-3) of the crassostrea gigas has an atypical CRD (carbohydrate recognition domain) and only contains four beta-sheets and one disulfide bond. The recombinant protein has a growth inhibition effect on Gram-negative bacterium escherichia coli and gram-positive bacterium staphylococcus aureus, has a destructive effect on cells of staphylococcus aureus, and has potential application value in the aspect of development of an antibacterial drug, a novel immune preparation, a feed additive and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to the application of a recombinant protein of long oyster C-type lectin-3 (CgCLec-3). Background technique [0002] C-type lectins are an important class of pattern recognition receptors that play an important role in innate immune responses. The carbohydrate recognition domain (CRD) is an important functional region of C-type lectins, which recognizes and binds glycoproteins in a calcium-dependent manner. A typical CRD usually consists of about 130 amino acids in a long bicyclic structure. The upper part of the structure consists of four β-sheets, while the lower part contains two α-helices and four β-sheets. Four conserved Cys (C1–C4) form two disulfide bonds at the base of the ring. Studies have shown that in the innate immune response, C-type lectins not only participate in the recognition and binding of invading pathogens as pattern recognition molecul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/57A61P31/04
CPCY02A50/30
Inventor 宋林生李慧张峘刘瑞贾志浩王玲玲
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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