Hybridoma cell, monoclonal antibody for anti-human cyclophilin A and application thereof

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

Active Publication Date: 2018-11-27
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 for anti-human cyclophilin A and application thereof
  • Hybridoma cell, monoclonal antibody for anti-human cyclophilin A and application thereof
  • Hybridoma cell, monoclonal antibody for anti-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 Use high-purity recombinant human CypA protein as the immunogen and completely mix it with complete Freund's adjuvant or incomplete Freund's adjuvant to immunize BALB / c mice subcutaneously. The inoculation is carried out every 2 to 4 weeks, and booster 3 ~4 times until the antibody titer of the inoculated antigen in the immunized animal is fully increased to 1:50000. The antibody titer in tail blood serum of immunized mice was determined by ELISA method. A final booster immunization was given 4 days before fusion.

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

[0047] 1.3 Selective culture of fused cells with HAT selective medium to kill unfused myeloma cells. At the same t...

Embodiment 2

[0051] Embodiment 2 Identification of anti-CypA mouse monoclonal antibody Ig subtype

[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 identification was carried out according to the steps using the collected cell-secreted antibody supernatant. The experimental results are attached figure 1 Shown: The obtained immunoglobulin is IgG1 subtype, and the light chain is κ 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 of hybridoma cells was extracted, and a commercially available reverse transcriptase cDNA synthesis kit was used, and the obtained cDNA was used as a template to perform PCR amplification using universal primers for mouse light chain and heavy chain variable region genes. The primers for the upstream end of the light chain variable region of the mouse immunoglobulin gene used 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 light chain variable region of the mouse immunoglobulin gene are:

[0061] 1121-1...

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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 specifically binding to human cyclophilin A (CypA) protein, a hybridoma cell strain producing the antibody, and the antibody light and heavy chain variable region genes and corresponding genes The encoded amino acid sequence. The antibody is used, for example, to detect and quantify the expression level of CypA in cells and tissues, and to prepare drugs for diagnosis or treatment of tumors and inflammations. Background technique [0002] Cyclophilin A, also known as cyclophilin protein A (hereinafter referred to as CypA), is the main member of the cyclophilin protein family, with a molecular weight of about 18 kDa. CypA is the main receptor of the immunosuppressive drug cyclosporine (Cyclosporine A, CsA) in cells, and the complex formed by the two can inhibit the phosphatase activity of calcineurin, thereby preventing the activation of T cell nuclear factor (NF- ATs) translocation into the nucleu...

Claims

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Application Information

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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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