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Hybridoma cell, monoclonal antibody against human cyclophilin a and application thereof

A monoclonal antibody and hybridoma cell technology, applied in the direction of anti-enzyme immunoglobulin, antibody, application, etc., can solve the problem of lack of effective technical means for cancer

Active Publication Date: 2021-03-16
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Cancer is one of the major diseases that plague human health around the world. Currently, there is still a lack of effective technical means for the diagnosis and treatment of cancer
At present, there is no monoclonal antibody or peptide against CypA protein as a drug for cancer diagnosis or treatment.
However, there is no report on the use of monoclonal antibodies or peptide drugs directly targeting CypA for the treatment of rheumatoid arthritis or other inflammation-related diseases
[0005] To sum up, CypA is a new broad tumor marker that has received attention in recent years. At the same time, CypA also plays a key role in the progression of inflammatory diseases. At present, there is no monoclonal antibody diagnosis against CypA protein at home and abroad. and therapeutic drugs

Method used

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  • Hybridoma cell, monoclonal antibody against human cyclophilin a and application thereof
  • Hybridoma cell, monoclonal antibody against human cyclophilin a and application thereof
  • Hybridoma cell, monoclonal antibody against human cyclophilin a and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Preparation of mouse anti-human Cyclophilin A protein monoclonal antibody

[0045] 1.1 Using high-purity recombinant human CypA protein as the immunogen and complete Freund's adjuvant or incomplete Freund's adjuvant, the BALB / c mice were injected subcutaneously to immunize BALB / c mice. ~4 times, until the antibody titer of the vaccinated antigen in the immunized animal is fully increased to 1:50000. Antibody titers in tail blood serum of immunized mice were determined by ELISA. The last booster 4 days before fusion.

[0046] 1.2 According to the commonly used method, such as the method described in "Monoclonal Antibody Preparation Technology", spleen cells of the immunized mouse were fused with the myeloma cell line SP2 / 0, and the fusion agent was 50% PEG.

[0047] 1.3 The fusion cells were selectively cultured in HAT selective medium to kill the unfused myeloma cells. At the same time, indirect ELISA was used to screen hybridoma cells with positive express...

Embodiment 2

[0051] Example 2 Identification of Ig subtypes of anti-CypA mouse monoclonal antibodies

[0052] The immunoglobulin subtype identification of the anti-CypA mouse monoclonal antibody obtained in Example 1 was carried out according to the instructions of the commercially available mouse monoclonal antibody subtype identification kit (PIERCE), and the collected cell-secreted antibody supernatant was used for identification according to the steps. The experimental results are attached figure 1 Shown: The immunoglobulin obtained is of the IgG1 subtype and the light chain is the kappa chain.

Embodiment 3

[0053] Example 3 Identification of heavy chain and light chain variable region genes of anti-CypA mouse monoclonal antibody

[0054] Total RNA from hybridoma cells was extracted, and a commercial reverse transcriptase cDNA synthesis kit was used. The obtained cDNA was used as a template, and PCR amplification was performed using universal primers for mouse light chain and heavy chain variable region genes. The primers used for the upstream end of the variable region of the light chain of the mouse immunoglobulin gene are:

[0055] LL1: 5'-GGGGATATCCACCATGGAGACAGACACACTCCTGCTAT-3';

[0056] LL2: 5'-GGGGATATCCACCATGGATTTTCAAGTGCAGATTTTCAG-3';

[0057] LL3: 5'-GGGGATATCCACCATGGAG(AT)CACA(GT)(AT)CTCGGGTCTTT(GA)TA-3';

[0058] LL5: 5'-GGATATCCACCATG(GT)CCCC(AT)(AG)CTCAG(CT)TC(CT)CT(TG)GT-3';

[0059] MVK: 5'-GGGGATATCCACCATGAAGTTGCCTGTTAGGCTGTTG-3';

[0060] The primers for the downstream end of the variable region of the light chain of the mouse immunoglobulin gene are:

[0061...

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Abstract

The inventio discloses a hybridoma cell, a monoclonal antibody for anti-human cyclophilin A and an application thereof. The invention adopts a traditional cell hybridoma preparation technique for acquiring a hybridoma cell strain for stably secreting the monoclonal antibody of the anti-human cyclophilin A (cyclophilin A, CypA). The preservation number of the hybridoma cell is CCTCC NO: C201126; the secreted antibody can specifically identify CypA protein molecules expressed in various tissues of a human body; and a new effective tool is provided for diagnosing and treating cancers and inflammations.

Description

technical field [0001] The present invention relates to a monoclonal antibody that specifically binds to human cyclophilin A (Cyclophilin A, CypA) protein and a hybridoma cell line producing the antibody, as well as the variable region genes of light and heavy chains of the antibody and corresponding genes the encoded amino acid sequence. The antibodies are used, for example, in the detection and quantification of CypA expression levels in cells and tissues, and in the preparation of medicaments for the diagnosis or treatment of tumors and inflammation. Background technique [0002] Cyclophilin A, also known as cyclophilin A (hereinafter referred to as CypA), is the main member of the cyclophilin family with a molecular weight of about 18kDa. CypA is the main intracellular receptor of the immunosuppressive drug Cyclosporine A (CsA), and the complex formed by the two can inhibit the phosphatase activity of calcineurin, thereby preventing the activation of T cell nuclear fact...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/40C12N15/13G01N33/577G01N33/574G01N33/573A61K39/395A61P35/00A61P29/00
CPCA61P29/00A61P35/00C07K16/40C07K2317/56C07K2317/565C07K2317/76G01N33/573G01N33/57484G01N33/577G01N2333/99
Inventor 陈志南张征杨向民边惠洁吴佼朱平张阳
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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