Crassostrea gigas DM9 structural domain-containing recombinant protein rCgDM9CP-6, and preparation method and application thereof

A technology of recombinant protein, long oyster, applied in the field of molecular biology

Pending Publication Date: 2021-07-30
DALIAN OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, so far, there is no information about the recombinant protein rCgDM9CP-6 containing the DM9 domain of the long oyster, the preparation method and its function as an inhibitor of Gram-positive bacteria. M. luteus Drug-related reports

Method used

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  • Crassostrea gigas DM9 structural domain-containing recombinant protein rCgDM9CP-6, and preparation method and application thereof
  • Crassostrea gigas DM9 structural domain-containing recombinant protein rCgDM9CP-6, and preparation method and application thereof
  • Crassostrea gigas DM9 structural domain-containing recombinant protein rCgDM9CP-6, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0036] Experimental example 1: Detection of the binding activity of the recombinant protein rCgDM9CP-6 containing the DM9 domain of the oyster of the present invention to bacteria

[0037] Based on the western blotting method, the recombinant protein rCgDM9CP-6 was detected against two Gram-negative bacteria (Vibrio brilliant and Escherichia coli), two Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and two fungi. Saccharomyces rovia and Pichia pastoris binding activity. The sources of the strains used are as follows: Vibrio splendidus ( Vibrio splendidus JZ6) was purchased from Beijing Microorganism Culture Collection Center, Escherichia coli ( Escherichia coli ) was purchased from Beijing Quanshijin Company, Staphylococcus aureus ( Staphylococcus aureus ) was purchased from Beijing Microorganism Culture Collection Center, Micrococcus luteus ( Micrococcus luteus ) was purchased from Beijing Microorganism Culture Collection Center, Yarrowia sacchar...

experiment example 2

[0057] Experimental Example 2: Detection of sugar-binding activity of the recombinant protein rCgDM9CP-6 containing the DM9 domain of oyster of the present invention

[0058] The enzyme-linked immunosorbent assay (ELISA) test was used to detect the combination of the recombinant protein rCgDM9CP-6 containing the DM9 domain of the present invention with various PAMPs. The four sugars used, D-Mannose, Mannose, LPS and PGN, were purchased from Sigma.

[0059] The specific operation steps are as follows:

[0060] (1) Na 2 CO 3 with NaHCO 3 Prepare the coating solution with a pH value of 7.6 according to the concentration of 15 mmol / L and 35 mmol / L, and dissolve the four sugars D-Mannose, Mannose, LPS and PGN to adjust the concentration to 125 μg / mL, and then add 100 μL, refrigerate overnight at 4°C;

[0061] (2) Discard the coating liquid and wash with TBS-T 4 times, 4 min each time;

[0062] (3) After washing, add 250 μL of 3% BSA to the wells and block them in a constant t...

experiment example 3

[0072] Experimental example 3: detection of phagocytosis-promoting activity of recombinant protein rCgDM9CP-6 containing DM9 domain in long oyster of the present invention

[0073] Two microorganisms (Vibrio splendidus and Staphylococcus aureus) were labeled with fluorescein isothiocyanate isomer (FITC), and then the phagocytic efficiency of oyster hemolymphocytes was detected by flow cytometry.

[0074] The strain source is as above.

[0075] The specific operation is as follows:

[0076] (1) Cultivate the above two microorganisms separately and collect the bacteria;

[0077] (2) Use formaldehyde to mix with microorganisms and fix for 10 min;

[0078] (3) Collect bacteria by centrifugation at 4,000 rpm for 10 min; 0.1 M NaHCO 3 After washing three times, incubate in 0.1 M NaHCO containing 0.1 mg / mLFITC 3 Incubate at 25°C for 2 h with gentle shaking;

[0079] (4) After centrifuging and discarding the supernatant, use TBS buffer to wash the microorganisms until they are co...

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Abstract

The invention discloses a crassostrea gigas DM9 structural domain-containing recombinant protein rCgDM9CP-6. The amino acid sequence of the crassostrea gigas DM9 structural domain-containing recombinant protein rCgDM9CP-6 is as shown in SEQ ID NO. 1; A preparation method of the recombinant protein rCgDM9CP-6 sequentially comprises the following steps: carrying out PCR amplification on a Crassostrea gigas CgDM9CP-6 gene coding region fragment by using primers P1 and P2, wherein the DNA sequence of the primer P1 is as shown in SEQ ID NO.2, and the DNA sequence of the primer P2 is as shown in SEQ ID NO.3; carrying out BamHI and Xhol I enzyme digestion on a PCR amplification product and a pET30a vector, then connecting and transforming through T4 ligase, and sequencing to identify a recombinant; and transferring the recombinant into an escherichia coli Transsetta (DE3) expression strain, performing induced culture, and then performing purification and renaturation to obtain the recombinant protein rCgDM9CP-6 with the amino acid sequence in the sequence table SEQ ID NO.1. The recombinant protein rCgDM9CP-6 can be applied to preparation of drugs for inhibiting gram-positive bacteria M.luteus.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to rCgDM9CP-6, a recombinant protein containing a DM9 domain of oyster, a preparation method and an application. Background technique [0002] long oysters ( Crassostrea gigas ) is an important marine cultured shellfish. Due to the lack of adaptive immune defense system of long oyster, it mainly relies on the innate immune system to resist the infection of exogenous pathogenic microorganisms. Due to the deterioration of the marine environment in recent years, various diseases caused by bacteria, fungi and viruses have continued to break out in oyster culture groups, causing huge economic losses. [0003] DM9 is a protein that contains a carbohydrate recognition domain (DM9) and can resist fungal and bacterial infections, promote cell phagocytosis and perform immune regulation functions. Studies have found that DM9 domain-containing proteins play an important...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70A61K38/17A61P31/04
CPCC07K14/43504C12N15/70A61P31/04A61K38/00
Inventor 王玲玲李奕男孙洁洁王伟林宋林生
Owner DALIAN OCEAN UNIV
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