Monoclonal antibody resisting XOD (xanthine oxidase) and preparation method and application thereof

A technology of xanthine oxidase and monoclonal antibody, which is applied in the field of pharmacy, medicine and immunology, can solve the problems of antibody difficulty and poor effect, and achieve the effect of enhancing immunogenicity, broad application prospect and important theoretical value

Inactive Publication Date: 2019-08-02
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The immune system uses the peptide-protein as a whole to stimulate an immune response, and the antibodies produced are directed against the peptide, the linker, and the carrier protein. Therefore, there are too many types of antibodies produced in the later stage, so that the screening needs In addition, the antibodies produced after coupling immunization with small molecular peptides such as B epitopes and KLH or BLH are mostly Th1 antibodies, while Th2 antibodies are less than Th1 antibodies, which are used to connect B cell epitopes or peptides The immunogen formed by hapten and the antibody used to prepare it are not suitable for or treat diseases with high Th1 (such as type 1 diabetes and its complications and related diseases, atherosclerosis, cardiovascular disease, senile dementia, kidney disease, nephritis , rheumatoid arthritis, reactive arthritis, multiple sclerosis, autoimmune thyroiditis, contact dermatitis, erythema nodosum, habitual abortion, etc.) Ineffective
[0006] The preparation of the monoclonal antibody of the present invention uses the xanthine oxidase B cell epitope as a hapten, coupled with the intramolecular adjuvant peptide prepared by our laboratory, and designs and synthesizes a multi-epitope peptide as an antigenic peptide or peptide immunogen, Immune spleen lymphocytes are obtained by immunizing biological preparations with such peptide immunogens. The intramolecular adjuvant consists of a 24-amino acid-specific polypeptide P277 peptide main Th2 epitope P2 peptide (448-460) at positions 437-460 of HSP60 The linear sequence at positions 626-630 of the proximal membrane region of JM2, which is the specific antigen of pancreatic islet cells-insulinoma-associated protein-2 (IA-2), is a B-cell epitope (626FEYQD630), named IA2 (5 ), by linking peptides, there is currently no report on the use of carrier peptides composed of P2 and IA2(5) to couple small peptides to prepare monoclonal antibodies and their uses

Method used

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  • Monoclonal antibody resisting XOD (xanthine oxidase) and preparation method and application thereof
  • Monoclonal antibody resisting XOD (xanthine oxidase) and preparation method and application thereof
  • Monoclonal antibody resisting XOD (xanthine oxidase) and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Preparation of peptide immunogen or antigenic peptide for immunization:

[0043]Connect the C-terminal of the IA2(5) peptide FEYQD (see SEQ ID NO.1 for the amino acid sequence) and the N-terminal of the Th2 epitope P2 peptide (see SEQ ID NO.2 for the amino acid sequence) to form a carrier through a flexible peptide or a connecting peptide The peptide, named IA2(5)-P2-1 (abbreviated as IP) and the B cell epitope O of human xanthine oxidase (indicated by B, O is a polypeptide sequence of xanthine oxidoreductase 1101RLEPYKKKNPS1111, a total of 11 amino acid residues, the amino acid sequence is shown in SEQ ID NO.3), and the C-terminus is covalently linked by a flexible peptide or a connecting peptide to form a peptide immunogen B-IA2(5)-P2-1 (this patent is O-IA2(5) )-P2-1, the amino acid sequence is shown in SEQ ID NO.4), the peptide immunogen or antigenic peptide is synthesized by FMOC solid-phase synthesis method, and the purity detected by HPLC is >90%. The...

Embodiment 2

[0044] Embodiment 2: Preparation of animal immunization and hybridoma cells

[0045] Animal immunization and preparation of immune spleen lymphocytes: the above antigenic peptides were emulsified with QuickAntibody-Mouse5W adjuvant, mixed with 0.9% saline for injection 1:1, and immunized 6-8 weeks old BALB / c mice by intramuscular injection. The immunization dose was 100 μg / rat, and the second immunization was carried out with the same immunization method and dose two weeks later. After two immunizations, the orbital blood was taken to determine the serum titer by ELISA gradient dilution, and the immunized spleen lymphocytes of the mouse with the highest antibody titer were selected for cell fusion. Conclusion: The above-mentioned antigenic peptides produced higher titers (greater than 1:10000) after immunizing mice, and could be used for the preparation of monoclonal antibodies ( figure 1 ).

[0046] Cell fusion: Sp2 / 0 derived from BALB / c mice was used for myeloma cells, and...

Embodiment 3

[0048] Example 3: Preparation and purification of monoclonal antibody mAb (named CPUKO1)

[0049] Preparation of mouse monoclonal antibody ascites: the method of producing monoclonal antibody in animals is adopted. Each Balb / c mouse was intraperitoneally injected with 0.5ml of sterilized liquid paraffin, and after 7 days, each mouse was intraperitoneally injected with 1×10 6 mouse hybridoma cells. After 7 days, observe the production of ascites in the mice. If the abdomen is obviously enlarged, the ascites can be extracted. Ascitic fluid contains red blood cells, cell debris, fibrin clots, and lipids. First remove the precipitated cells by low-speed centrifugation, then high-speed centrifugation to remove cell residues and small particles, and use a 0.2 μm microporous membrane to remove fibrin clots and lipids.

[0050] Caprylic acid-ammonium sulfate precipitation method: the initially purified ascitic fluid was crudely extracted with caprylic acid-ammonium sulfate precipit...

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Abstract

The invention relates to the related fields of immunology, pharmacy and medicine, discloses a method for preparing a hybridoma cell strain and a monoclonal antibody based on B cell epitope of XOD (xanthine oxidase) and application, and particularly relates to micromolecular antigen peptide which is quickly prepared by intramolecular carrier peptide coupling with B epitope of XOD via synthesizing.An immune mouse can produce strong immune response to prepare immune splenic lymphocytes and then prepare the hybridoma cell strain and the monoclonal antibody. The monoclonal antibody is secreted bythe hybridoma cell strain (collection number: CCTCC No.C201788), and is a Th2 type antibody; the monoclonal antibody is IgG1 subtype in the mouse, and can inhibit the activity of XOD; by specificallycombining with the XOD, an XOD detection reagent and an XOD inhibitor can be developed, so as to realize the effects of reducing the blood glucose, uric acid and creatinine, adjusting the oxidative stress and immune balance, enabling Th1 to tend to Th2, and relieving kidney inflammation. The monoclonal antibody can be prepared into medicines for preventing and treating the hypeluricemia, gout, kidney and hepatic disease, diabetes and complication, AS (ankylosing spondylitis), cardiovascular disease, higher Th1 and related diseases due to disorder of uric acid or oxidative stress.

Description

technical field [0001] The invention relates to the related fields of immunology, pharmacy and medicine. The present invention relates to anti-xanthine oxidase monoclonal antibody and its preparation method and application, especially relates to using the B cell epitope of human xanthine oxidase as a hapten, and especially relates to coupling with a small molecule intramolecular carrier peptide The B cell epitope peptide of human xanthine oxidase (XOD) is used to prepare multi-epitope peptide or antigenic peptide or peptide immunogen and obtain it quickly through synthesis. The peptide immunogen immunized mice can generate a strong immune response and can be used to prepare immunization Spleen lymphocytes, the immunized spleen lymphocytes can be used to prepare hybridoma cells to prepare monoclonal antibodies, or extract their genes through existing techniques to prepare various antibodies through antibody library technology or genetic engineering. The prepared monoclonal ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/40G01N33/573A61K39/395A61P19/06A61P3/10A61P13/12A61P1/16A61P9/10A61P25/28A61P27/02A61P9/00C12R1/91
CPCC07K16/40G01N33/573C07K2317/33C07K2317/76C07K2317/56C07K2317/565A61K2039/505G01N2333/9029
Inventor 李泰明顾小骞李雅刘晓然方金芝
Owner CHINA PHARM UNIV
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