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Application of Pacific oyster C-type lectin-2(CgCLec-2) recombinant protein with antibacterial activity

A technology of antibacterial activity and recombinant protein, applied in the field of molecular biology, can solve problems such as economic loss

Inactive Publication Date: 2015-04-01
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology can be useful for developing new treatments or preventing diseases caused by harmful microorganisms that may affect animals such as humans through their ability to recognize specific molecules called Oysters Lectins 2 protease which are involved with causing them's disease symptoms like staphylitis.

Problems solved by technology

This patented technical problem addressed in this patents relates to finding ways for fishermen to protect their crops from disease during harvesting periods due to poor adaptation between natural and artificial environments.

Method used

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  • Application of Pacific oyster C-type lectin-2(CgCLec-2) recombinant protein with antibacterial activity
  • Application of Pacific oyster C-type lectin-2(CgCLec-2) recombinant protein with antibacterial activity
  • Application of Pacific oyster C-type lectin-2(CgCLec-2) recombinant protein with antibacterial activity

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Experimental program
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Embodiment 1

[0021] The in vitro prokaryotic recombinant expression of the long oyster lectin-2 (CgCLec-2) gene coding region comprises the following steps:

[0022] 1. Construction of recombinant vector

[0023] The recombinant vector used in the present invention is pET-32a prokaryotic expression vector.

[0024] By PCR technology, gene-specific primers with specific restriction sites of Bam H I and Hind III were added at the 5' ends to amplify the coding region fragment of C-type lectin CgCLec-2 of oyster oyster:

[0025] Gene-specific primers are:

[0026] P1 (5'-CGCGGATCCATGGATACAGAAGTGACCTCAT-3'):

[0027] P2(5'-CCCAAGCTTCTATAAAGTTTGTTCACAGATG-3'),

[0028] 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 ...

Embodiment 2

[0051] Long oyster recombinant protein rCgCLec-2 recognizes and binds carbohydrates in a calcium-dependent manner

[0052] 20 μg of LSP, PGN and MAN were dissolved in 50 mM sodium carbonate-sodium bicarbonate buffer (pH 9.6), 100 μL per well was coated with a microtiter plate, and left overnight at 4°C. In order to avoid non-specific binding, 200 μL of 3% BSA was added and blocked for 1 h at 37 °C. Afterwards, 100 μL of 2-fold serially diluted recombinant protein rCgCLec-2 was added to each well, and the final concentration was 10 mM Ca 2+ or without adding Ca 2+ Incubate at 18°C ​​for 3 hours. Add 100 μL rat anti-rCgCLec-2 polyclonal antibody (1:1000) diluted with 3% BSA, and incubate at 37° C. for 1 h. Add 100 μL of HRP-labeled goat anti-rat IgG (1:3000), and incubate at 37°C for 1 hour. Wash 3 times with TBST between each incubation step, 5 min each time. After secondary antibody incubation and thorough washing, 100 μL TMB was added to each well, and after 30 minutes o...

Embodiment 3

[0054] Antibacterial activity of recombinant protein rCgCLec-2 from oyster oyster

[0055] 1. Culture and preparation of microorganisms

[0056] Use LB medium for Staphylococcus aureus at 37°C, 220rpm, and when it reaches the logarithmic growth phase, dilute the bacteria with Tris-HCl (50mmol L-1, pH=8.0) so that the number of colonies per milliliter of the bacteria solution is about It is 1×103CFU.

[0057] 2. Determination of antibacterial activity of recombinant protein CgCLec-2

[0058] Staphylococcus aureus in the logarithmic growth phase was collected by centrifugation and washed and resuspended with TBS (50mM Tris-Hcl, 150mM NaCl) (10 4 CFU). 50 μL of long oyster recombinant protein CgCLec-2 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. 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 cult...

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Abstract

The invention belongs to the technical field of molecular biology and in particular relates to application of a Pacific oyster C-type lectin-2(CgCLec-2) recombinant protein with the antibacterial activity. A Pacific oyster C-type lectin-2(CgCLec-2) gene is used for preparing a bacteriostatic agent or a feed additive. According to the application disclosed by the invention, a Pacific oyster C-type lectin-2(CgCLec-2) protein which is in vitro recombined and expressed has the effect of inhibiting growth of staphylococcus aureus of gram-positive bacteria and has potential application value on the aspects of developing anti-infective drugs, novel immune preparations and feed additives when used as en effective pattern recognition receptor.

Description

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Claims

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Application Information

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Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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