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Anti-human CXCL-2 monoclonal antibody 3-D3 as well as coding gene and application thereof

A technology of monoclonal antibody and coding gene, applied in application, antibody, genetic engineering, etc., can solve the problem of low antibody affinity

Pending Publication Date: 2021-02-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the existing CXCL-2 antibody has a low affinity, and there is still a lack of a high-affinity blocking CXCL-2 antibody

Method used

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  • Anti-human CXCL-2 monoclonal antibody 3-D3 as well as coding gene and application thereof
  • Anti-human CXCL-2 monoclonal antibody 3-D3 as well as coding gene and application thereof
  • Anti-human CXCL-2 monoclonal antibody 3-D3 as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: the construction of hybridoma cell line

[0053] 1. Materials

[0054] DNA immune adjuvant was purchased from Sigma; fetal bovine serum and PRMI1640 medium were purchased from Gibco; SP2 / 0 cells were purchased from ATCC; BALB / c mice were provided by the Experimental Animal Center of Zhejiang University.

[0055] 2. Method:

[0056] (1) Plasmid construction

[0057] Primers were designed according to the CXCL-2 gene sequence checked on NCBI, and the human cDNA was used as a template to amplify the CXCL-2 gene DNA sequence, which was constructed on the pcDNA3.1 plasmid by molecular biology methods.

[0058] (2) Immunization of mice

[0059] BALB / c mice aged 4-6 weeks were selected, and the constructed CXCL-2 gene recombinant plasmid was dissolved in PBS and an equal volume of adjuvant, mixed and emulsified, and subcutaneously and intraperitoneally multi-point immunized. Every 2 weeks after the first immunization, the same dose of plasmid mixed with adjuv...

Embodiment 2

[0066] Example 2: Antibody subclass identification and stability test

[0067] The subclass identification of the antibody was carried out according to the instructions of the antibody subclass identification kit, and the result was that the heavy chain was IgG1 type, and the light chain was Kappa type.

[0068] The hybridoma cell line was continuously subcultured in vitro for 3 months, and the antibody titer in the supernatant was determined; the frozen hybridoma cell line was recovered after 4 months, and the antibody titer in the supernatant was detected, and no significant changes occurred. Changes, indicating that the obtained antibody-producing hybridoma cell line has stable performance.

Embodiment 3

[0069] Example 3: Anti-CXCL-2 monoclonal antibody inhibits neutrophil migration experiment

[0070] 1. Materials

[0071] 2. Methods and Results

[0072] (1) Matrigel plate: Dilute it with BD Matrigel 1:8, coat the upper chamber surface of the bottom membrane of the Transwell chamber, place at 37°C for 30 minutes to polymerize the Matrigel into a gel, and hydrate the basement membrane before use.

[0073] (2) Preparation of cell suspension

[0074] (3) Cell inoculation

[0075] Add 100ul of decellularized suspension to the Transwell chamber, generally add 600ul of medium containing 20% ​​FBS to the lower chamber of a 24-well plate, and culture for 24 hours

[0076] (4) Results statistics. Take out the Transwell chamber, discard the culture medium in the well, wash twice with calcium-free PBS, fix with formaldehyde for 30 minutes, and air-dry the chamber properly. Stain with 0.1% crystal violet for 20 minutes, gently wipe off the upper layer of unmigrated cells with a cott...

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Abstract

The invention belongs to the technical field of bioengineering, and relates to an anti-human CXCL-2 monoclonal antibody 3-D3 as well as a coding gene and application thereof. The monoclonal antibody or a fragment thereof is combined with an extracellular region containing a chemotactic factor CXCL-2 and has an effect of blocking the function of the CXCL-2. The invention relates to the anti-human CXCL-2 monoclonal antibody, preparation of the anti-human CXCL-2 monoclonal antibody, a variable region amino acid sequence of the anti-human CXCL-2 monoclonal antibody, a gene coding sequence of the anti-human CXCL-2 monoclonal antibody and an application of the anti-human CXCL-2 monoclonal antibody. The monoclonal antibody capable of blocking the function of a human chemotactic factor CXCL-2 andthe encoding gene of the monoclonal antibody are newly found, the monoclonal antibody can be combined with an extracellular region of the human CXCL-2, combination of the CXCL-2 and a ligand of the CXCL-2 can be specifically blocked, signal transduction is inhibited, and thus the monoclonal antibody can be used as a blocking agent of a CXCL-2 signal channel. Therefore, the antibody becomes a novelantibody drug for tumor immunotherapy and treatment of acute and chronic infectious diseases and autoimmune diseases.

Description

technical field [0001] The invention belongs to the technical field of bioengineering. The monoclonal antibody or its fragment binds to the extracellular region containing the chemokine CXCL-2 and has the effect of blocking the function of CXCL-2. The invention relates to an anti-human CXCL-2 monoclonal antibody Cloned antibody and its preparation, variable region amino acid sequence and its gene coding sequence and application. Background technique [0002] Chemokines are a class of proteins that can regulate leukocyte migration and play an important role in inflammatory responses. In 1988, Cerami et al. purified and analyzed two heparin-binding proteins released by LPS-stimulated macrophages and discovered MIP-1 [Wolpe SD, Davatelis G, Sherry B, Beutler B, Hesse DG, Nguyen HT, et al. Macrophages secrete a novel heparin-binding protein with inflammatory and neutrophil chemokinetic properties. The Journal of experimental medicine. 1988; 167:570-81.] , and the following year...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24C12N15/13A61K39/395A61P31/00A61P35/00A61P37/00
CPCC07K16/24A61P31/00A61P35/00A61P37/00C07K2317/565C07K2317/92C07K2317/76A61K2039/505
Inventor 洪萌陈智朱海红任艳丽
Owner ZHEJIANG UNIV
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