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Human-mouse chimeric anti-CXCR2 full-molecule IgG and application thereof

A molecular and coding technology, applied in the field of biopharmaceuticals, can solve problems such as no combined reports

Active Publication Date: 2018-04-27
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Among the currently published antibodies targeting CXCR2, most are mainly for diagnosis and scientific research. There is no report that can bind to a specific amino acid region in the extracellular domain of CXCR2, and can also inhibit or block the binding function of CXCR2.

Method used

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  • Human-mouse chimeric anti-CXCR2 full-molecule IgG and application thereof
  • Human-mouse chimeric anti-CXCR2 full-molecule IgG and application thereof
  • Human-mouse chimeric anti-CXCR2 full-molecule IgG and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0023] 1. Preparation of mouse-derived anti-CXCR2 hybridoma cells

[0024] 2. Screening, preparation and identification of murine anti-CXCR2 antibody

[0025] 3. Preparation, expression and purification of human-mouse chimeric anti-CXCR2 whole molecule IgG

[0026] 4. Characteristic analysis of human-mouse chimeric anti-CXCR2 whole molecule antibody

[0027] 5. Detection of the competitive inhibitory effect of anti-CXCR2 whole molecule IgG on GROα protein

Embodiment 1

[0028] Example 1. Preparation of mouse-derived anti-CXCR2 hybridoma cells

[0029] According to the human CXCR2 gene (UniProtKB P25025) extracellular region sequence (A286-A297), CERRNHIDRALD-C was designed as an antigen with a specific epitope, synthesized by Nanjing GenScript Biotechnology Co., Ltd., 5 mg (95% purity), and coupled to OVA and KLH. The short peptide coupled with OVA is an antigen for immunization, and the short peptide coupled with KLH is an antigen for screening and detection.

[0030] CXCR2 extracellular region recombinant protein was used as the immunogen to immunize the abdomen of pure BALB / c mice by subcutaneous injection, 100 μg / ml each time, five times in total, and intraperitoneal booster immunization was carried out three days before the last immunization. On the day of fusion, the mouse spleen was taken, and a single-cell suspension was prepared with DMEM medium (GIBCO, USA). In the presence of 50% PEG (PH 8.0), the spleen cells and SP2 / 0 mouse myel...

Embodiment 2

[0031] Example 2. Screening, preparation and identification of murine anti-CXCR2 antibodies

[0032] The enzyme-linked immunoassay (ELISA) detection and screening was carried out according to the growth condition of the hybridoma cells, and the specific method is as follows. The cells in the positive wells were cloned and cultured again. After 6 times of subcloning, when all the monoclonal cells in the wells were positive for CXCR2-KLH and negative for KLH, several wells were taken for expansion and cultured and partially frozen.

[0033] The ELISA method is used to screen CXCR2-positive and normal mouse serum-negative monoclonal antibodies. The specific method is as follows:

[0034] (1) The chemically synthesized CXCR2-KLH polypeptide was coated with ELISA on a 96-well plate, diluted to 20 μg / ml with coating solution (0.1M carbonate buffer, pH9.6), 100 μl was added to each well, and overnight at 4°C;

[0035] (2) Wash 5 times with PBST washing solution (PBS containing 0.05%...

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Abstract

The invention discloses a human-mouse chimeric anti-CXCR2 full-molecule IgG and an application thereof and belongs to the field of biological pharmacy. The human-mouse chimeric anti-CXCR2 full-molecule IgG comprises a heavy chain variable region and a light chain variable region and is characterized in that the light chain variable region has a nucleic acid sequence shown in the formula of SEQ IDNO. 1 and the heavy chain variable region has a nucleic acid sequence shown in the formula of SEQ ID NO. 2. The mouse immunized by the specific recombinant CXCR2 protein is used, a mouse-derived anti-CXCR2 monoclonal antibody is prepared through a hybridoma technology, and the recombinant human-mouse chimeric anti-CXCR2 full-molecule IgG is prepared through genetic engineering and antibody engineering techniques. The chimeric antibody can effectively recognize the CXCR2 extracellular domain amino acid fragment and inhibit the binding of the CXCR2 protein to the GRO alpha protein.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a human-mouse chimeric anti-CXCR2 full-molecule IgG antibody, and also relates to the DNA molecule, expression vector, host cell and application of the above-mentioned full-molecule IgG antibody. Background technique [0002] Chemokine is a general term for cytokines that can cause cells to undergo chemotaxis. It was first discovered in mammals, birds and fish animals. Chemokines are secreted by various cells such as neutrophils and monocytes, and are mainly expressed on the surface of inflammatory cells such as neutrophils and macrophages, and can also be expressed on epithelial cells, smooth muscle cells and fibroblasts, etc. cell. According to the different characteristics of the primary structure of the polypeptide chain, chemokines are divided into CXC subfamily and CC subfamily. The CXC family of chemokines has an ELR (glutamic acid-leucine-arginine) structure, which can p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCA61K2039/505C07K16/2866C07K2317/24C07K2317/56C07K2317/76C07K2317/92
Inventor 唐奇冯振卿朱进黄骁辰贾立周
Owner NANJING MEDICAL UNIV
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