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Anti-dipeptidyl peptidase 4 monoclonal antibody, and preparation method and application thereof

A technology of dipeptidyl peptidase and monoclonal antibody, which is applied in immunology, pharmacy and medical related fields, can solve problems such as poor effect and antibody difficulty, and achieve enhanced immunogenicity, simple screening of hybridoma cells, and important theory value effect

Inactive Publication Date: 2019-08-16
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, so the antibodies produced are directed against the peptide, some against the linker, and some against 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 the treatment of diseases with high Th1 (such as type 1 diabetes and its complications and related diseases, atherosclerosis, cardiovascular disease, Alzheimer's disease, 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 B cell epitope of DPP-4 as a hapten, coupled with the intramolecular adjuvant peptide prepared in our laboratory, and designs and synthesizes a multi-epitope peptide as an antigenic peptide or peptide immunogen, Immune spleen lymphocytes are obtained by direct immunization of 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 ) and islet cell-specific antigen-insulinoma-associated protein (Insulinoma associated protein-2, IA-2), the linear sequence at position 626-630 of the proximal membrane region of JM2 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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  • Anti-dipeptidyl peptidase 4 monoclonal antibody, and preparation method and application thereof
  • Anti-dipeptidyl peptidase 4 monoclonal antibody, and preparation method and application thereof
  • Anti-dipeptidyl peptidase 4 monoclonal antibody, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Preparation of peptide immunogen or antigenic peptide for immunization:

[0045]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 Peptide, named IA2(5)-P2-1 (abbreviated as IP) and human DPP-4 B cell epitope D41 (indicated by B, D41 is a polypeptide sequence 88VFLENSTFDE97 of DPP-4, a total of 10 amino acid residues base, 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 D41-IA2 (5)-P2 -1, the amino acid sequence is shown in SEQID 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 B cell epitope D41 used for analy...

Embodiment 2

[0046] Embodiment 2: Preparation of animal immunization and hybridoma cells

[0047] 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 blood was taken from the orbit, and the serum titer was determined 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 ).

[0048] Cell fusion: Sp2 / 0 derived from BALB / c mice was used for myel...

Embodiment 3

[0050] Example 3: Preparation and purification of monoclonal antibody mAb (named CPU-KD4)

[0051] 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 particulate matter, and use a 0.2 μm microporous membrane to remove fibrin clots and lipids.

[0052] Caprylic acid-ammonium sulfate precipitation method: the ascites that was initially purified was crudely extracted with caprylic acid-ammonium sul...

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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 line and a monoclonal antibody based on B cell epitopes of dipeptidyl peptidase 4 (DPP-4) and an application thereof, and especially, relates to small molecular antigen peptides prepared by coupling the B epitopes of DPP-4 with intramolecular carrier peptides and quickly obtained by a synthetic method; the small molecular antigen peptides immunize mice to produce strong immune response, and can be used for preparing immune spleen lymphocytes and preparing hybrid tumor cells, antibodies and the like. The monoclonal antibody is secreted by the hybridoma cell line (having the preservation number of CCTCC No:C2017227), is a Th2 antibody, is IgG1 in the mice, inhibits the enzyme activity of DPP-4, is specifically combined with DPP-4, can develop a DPP-4 detection reagent and a DPP-4 inhibitor, can significantly reduce blood sugar, uric acid and four indexes ofcreatinine blood lipid, increases GLP-1, regulates oxidative stress and immune balance from Th1 trending to Th2, and reduces pancreatic inflammation; prepared drugs are used for preventing and treating diabetes and complications thereof, hyperuricemia, gout, kidney and liver diseases, hyperlipidemia, AS, cardiovascular diseases and Th1-related diseases.

Description

technical field [0001] The invention relates to the related fields of immunology, pharmacy and medicine. The present invention relates to anti-dipeptidyl peptidase 4 (Dipeptidyl peptidase 4, DPP-4) monoclonal antibody and its preparation method and application, particularly relates to the B cell epitope of human dipeptidyl peptidase 4 as a hapten, and especially Involves the preparation of multi-epitope peptides or antigenic peptides or peptide immunogens by coupling the B-cell epitope peptide of human dipeptidyl peptidase 4 (DPP-4) with a small molecule intramolecular carrier peptide and obtains it quickly by synthesis , Peptide immunogen immunized mice can produce a strong immune response, which can be used to prepare immunized spleen lymphocytes, which can be used to prepare hybridoma cells to prepare monoclonal antibodies or extract their genes through existing techniques through antibody library technology or gene Engineering to produce a variety of antibodies. The prep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N5/20G01N33/573G01N33/577A61K39/395A61P1/16A61P3/06A61P3/10A61P9/10A61P13/12A61P19/06A61P25/28A61P27/02
CPCC07K16/40G01N33/573G01N33/577C07K2317/56A61K2039/505
Inventor 李泰明顾小骞焦瑞方金芝
Owner CHINA PHARM UNIV
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