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Application of crassostrea gigas C-type agglutinin-4 (CgCLec-4) recombination protein

A technology of recombinant protein and lectin, applied in the field of molecular biology, can solve problems such as the death of long oysters and the economic loss of long oyster aquaculture

Inactive Publication Date: 2016-06-22
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the deteriorating marine environment in recent years, large-scale deaths of oysters have occurred frequently, and various microbial diseases in seawater have caused huge economic losses to the aquaculture industry of oysters.

Method used

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  • Application of crassostrea gigas C-type agglutinin-4 (CgCLec-4) recombination protein
  • Application of crassostrea gigas C-type agglutinin-4 (CgCLec-4) recombination protein
  • Application of crassostrea gigas C-type agglutinin-4 (CgCLec-4) recombination protein

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Experimental program
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Effect test

Embodiment 1

[0017] Long oyster recombinant protein rCgCLec-4 (Genbank number: XP_011415552) recognizes and binds sugars and microorganisms in a calcium-dependent manner

[0018] 1. Construction of recombinant protein

[0019] Obtain the full length of CgCLec-4 gene from NCBI, use gene-specific primers, clone CgCLec-4 according to ExTaq DNA polymerase instructions, and connect the cloned fragment to the cloning vector pMD19-T. Select the cloned strain with correct sequencing, select the appropriate restriction site, and add it to the 5' end of the gene-specific primer to amplify the target fragment with the restriction site, using specific restriction endonuclease Carry out double enzyme digestion on the target fragment. Use T4 ligase (NEB) to connect the double-digested target fragment and the expression vector, and transform the ligated recombinant expression plasmid into the clone-competent TOP10. The successfully constructed recombinant expression plasmids were transformed into expre...

Embodiment 2

[0030] Antibacterial activity of recombinant protein rCgCLec-4 from oyster oyster

[0031] 1. Culture and preparation of microorganisms

[0032] Escherichia coli and Staphylococcus aureus use LB medium at 37°C, 220rpm, when they reach the logarithmic growth phase, use Tris-HCl (50mmolL -1 , pH=8.0) to dilute the bacteria so that the number of colonies per milliliter of bacteria liquid is about 1×10 4 CFU.

[0033] Vibrio vulnificus, Vibrio alginolyticus and Pseudomonas aeruginosa use 2216E medium at 28°C, 140rpm, when culturing to logarithmic growth phase, use Tris-HCl (50mmolL -1 , pH=8.0) to dilute the bacteria so that the number of colonies per milliliter of bacteria liquid is about 1×10 4 CFU.

[0034] 2. Determination of antibacterial activity of recombinant protein CgCLec-4

[0035] The five kinds of bacteria in the logarithmic growth phase were collected by centrifugation and washed with TBS (50mM Tris-Hcl, 150mMNaCl) and resuspended (10 4 CFU). 50 μL of the recomb...

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Abstract

The invention belongs to the technical field of molecular biology and particularly relates to application of crassostrea gigas C-type agglutinin-4 (CgCLec-4).According to the method, an in vitro recombination expression technology is utilized, recombination protein of a carbohydrate recognition domain (CRD) of crassostrea gigas CgCLec-4 is obtained, and application of crassostrea gigas C-type agglutinin-4 (CgCLec-4) recombination protein to preparation of a bacteriostatic agent or a fodder additive is obtained.The obtained recombination protein can be used for development of an antibacterial fodder additive or novel immunopotentiator.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to the application of C-type lectin-4 (CgCLec-4) of long oyster. Background technique [0002] The long oyster is widely distributed in the world and is an important marine cultured species. Due to the deteriorating marine environment in recent years, large-scale deaths of oysters have occurred frequently, and various microbial diseases in seawater have caused huge economic losses to the aquaculture industry of oysters. Therefore, it is of great significance to carry out basic research on the immune defense mechanism of oysters to support the healthy and sustainable development of the aquaculture industry. At the same time, as a representative of molluscs, the long oyster has a relatively primitive evolutionary status, which is of great significance to the study of invertebrate immunology and comparative immunology. [0003] C-type lectins are a class of prot...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K35/618A61P31/04A23K20/147
CPCA61K35/618A61K38/1767
Inventor 王玲玲贾志浩张峘宋林生
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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