Recombinant crassostrea gigas high-mobility family protein r-CgHMGB1, preparation method and application thereof

A high-mobility, r-cghmgb1 technology, applied in the field of molecular biology, can solve problems such as antibacterial and immune functions of high-mobility group proteins in oysters that have not been seen.

Active Publication Date: 2022-01-11
DALIAN OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Purification of HMGB1 from human and rat testis has antibiotic-like function and bactericidal activity against several types...

Method used

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  • Recombinant crassostrea gigas high-mobility family protein r-CgHMGB1, preparation method and application thereof
  • Recombinant crassostrea gigas high-mobility family protein r-CgHMGB1, preparation method and application thereof
  • Recombinant crassostrea gigas high-mobility family protein r-CgHMGB1, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0036] Experimental Example 1: Recombinant oyster high mobility group protein r- C g HMGB1 bacterial binding activity detection

[0037] Detection of recombinant long oyster high mobility group protein r- based on Western blotting C g HMGB1 against two Gram-negative bacteria: Vibrio splendidus ( Vibrio splendidus JZ6 , abbreviated as V. splendidus group, the same below) and Escherichia coli ( Escherichia coli , E. coli ), three Gram-positive bacteria: Micrococcus luteus ( Micrococcus luteus , M. luteus ), Staphylococcus aureus ( Staphylococcus aureus , S. aureus ) and Bacillus subtilis ( Bacillus subtilis , B. subtilis ) and a fungus Pichia pastoris ( Pichia pastoris GS115 , P. pastoris ) binding activity. The sources of the strains used are as follows: Vibrio resplendent was purchased from Beijing Microorganism Culture Collection Center, Escherichia coli was purchased from Beijing Quanshijin Company, Staphylococcus aureus was purchased from Beijing M...

experiment example 2

[0057] Experimental Example 2: Recombinant oyster high mobility group protein r- C g Carbohydrate binding activity assay of HMGB1

[0058] Detection of recombinant high mobility group protein r- C g Binding of HMGB1 to various PAMPs. The peptidoglycan (PGN), lipopolysaccharide (LPS) and glucan (GLU) used were purchased from Sigma.

[0059] The specific operation steps are as follows:

[0060] (1) Dissolve 10 μg of various PAMPs in Na 2 CO 3 with NaHCO 3 According to 15 mmol·L -1 and 35 mmol·L -1 Concentration configuration of the coating solution with a pH value of 7.6, 100 μL per well to coat the microtiter plate, and refrigerate overnight at 4 °C;

[0061] (2) Discard the coating liquid and wash 3 times with TBST, 5 min each time;

[0062] (3) After washing, add 200 μL of 3% BSA to the wells and seal them in a constant temperature incubator at 37°C for 1 h;

[0063] (4) Wash with TBST 3 times after sealing, 5 min each time;

[0064] (5) Add 100 μL of diluted conce...

experiment example 3

[0072] Experimental Example 3: Recombinant oyster high mobility group protein r- C g Bacteriostatic activity assay of HMGB1

[0073] Recombinant oyster high mobility group protein r- C g HMGB1 was incubated with two kinds of bacteria (Vibrio splendidus and Escherichia coli), and the concentration of the bacterial solution was detected using a microplate reader (Tecan Infinite M1000 PRO), and the recombinant long oyster high mobility group protein r- C g Bacteriostatic activity of HMGB1.

[0074] The strain source is as above.

[0075] The specific operation is as follows:

[0076] (1) This experiment is divided into three groups: the embodiment of the present invention recombinant long oyster high mobility group protein r- C g HMGB1 treatment group, rTrx control group (negative control), TBS control group (blank control), the number of repetitions of each experimental group is not less than three parallel;

[0077] (2) Cultivate the above two microorganisms separately and...

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Abstract

The invention discloses a recombinant crassostrea gigas high-mobility family protein r-CgHMGB1, a preparation method and application thereof. The amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1. The preparation method is carried out according to the following steps in sequence: carrying out PCR amplification on a crassostrea gigas CgHMGB1 coding region fragment by using primers P1 and P2, wherein the DNA sequence of the primer P1 is shown as SEQ ID NO. 2, and the DNA sequence of the primer P2 is shown as SEQ ID NO. 3; carrying out BamHI and XholI enzyme digestion on a PCR amplification product and a pET30a vector, then connecting through T4 ligase, and sequencing and identifying a recombinant; transferring the recombinant into an escherichia coli Transetta (DE3) expression strain for induction culture, and then purifying and renaturing. The recombinant crassostrea gigas high-mobility family protein r-CgHMGB1 can be applied to preparation of medicines for resisting vibrio splendidus and escherichia coli.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a recombinant long oyster high mobility group protein r- C g HMGB1, preparation method and application thereof. Background technique [0002] High mobility group box B1 (HMGB1) is a class of molecules containing high mobility group box (HMG) domain and highly conserved amino acid sequence, which plays an important role in antibacterial immunity and inflammatory response. This domain was first extracted and identified in bovine thymus in 1973. In recent years, more and more HMG domain-containing proteins have been found in vertebrates and invertebrates. HMGB1 contains two HMG domains called A-box and B-box and a carboxy-terminal acidic tail region, B-box is a functional domain that causes inflammation, and A-box has a certain antagonistic effect on B-box , residues 201–205 in the carboxy-terminal acidic tail region are responsible for the antibacterial activity of HMGB...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/70C12N15/66A61K38/17A61P31/04
CPCC07K14/43504C12N15/70C12N15/66A61P31/04C12N2800/101A61K38/00Y02A50/30
Inventor 孙洁洁吕晓倩王玲玲宋林生
Owner DALIAN OCEAN UNIV
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