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VHH (variable domain of heavy chain of heavy-chain antibody) antibody gene derived from anti-CyPA (CyclophilinA) animal of family Camelidae, encoded polypeptide, and application thereof

A gene and animal technology, applied to the gene and polypeptide of alpaca heavy chain VHH antibody, the application field of the gene and polypeptide can solve the problems that antibody drugs have not been reported

Active Publication Date: 2014-04-16
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, there are no reports of antibody drugs targeting CypA

Method used

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  • VHH (variable domain of heavy chain of heavy-chain antibody) antibody gene derived from anti-CyPA (CyclophilinA) animal of family Camelidae, encoded polypeptide, and application thereof
  • VHH (variable domain of heavy chain of heavy-chain antibody) antibody gene derived from anti-CyPA (CyclophilinA) animal of family Camelidae, encoded polypeptide, and application thereof
  • VHH (variable domain of heavy chain of heavy-chain antibody) antibody gene derived from anti-CyPA (CyclophilinA) animal of family Camelidae, encoded polypeptide, and application thereof

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Experimental program
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Embodiment Construction

[0017] (1) Screening of anti-CypA heavy chain VHH antibody A1

[0018] 1. Immunity of alpacas

[0019] Two alpacas were immunized with CypA, and the immunization scheme was as follows: Alpaca antigen dosage for immunization: 0.5mg, 0.5mg, 1.0mg, 1.0mg, 2.0mg, 4.0mg, 4.0mg. The interval between immunizations was 2 weeks. Method: The first immunization was to grind and mix the antigen plus Freud's complete adjuvant, and inject it subcutaneously at multiple points. Subsequent immunization, antigen plus Freud's incomplete adjuvant, multi-point subcutaneous injection, 0.5ml / point, 4 points / time. Take a small amount of blood before each immunization, and use the ELISA method to detect the titer of anti-CypA antibody in alpaca blood, such as figure 1 As shown, after 7 times of immunization, the antibody titer in the blood of one alpaca reached 1:186,000.

[0020] 2. Construction of anti-cyclophilin A immune phage antibody library

[0021] The peripheral blood of immunized alpaca...

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Abstract

The invention relates to VHH (variable domain of heavy chain of heavy-chain antibody) antibody gene derived from anti-CyPA (CyclophilinA) animal of family Camelidae, encoded polypeptide, and application of the gene and the polypeptide in preparing drugs for treating inflammatory diseases such as rheumatoid arthritis. The VHH antibody gene has gene sequence shown in sequence table (400)1, the encoded polypeptide has amino acid sequence shown in sequence table (400)2. Lama pacos as immune animal is used, and phage display techniques (PDT) are adopted to successfully obtain an anti-CyPA VHH antibody capable of specifically combining with expressed CyPA antigen. The antibody can inhibit monocytes THP-1 chemotaxis and gelatinase secretion induced by CyPA. Dimer or polymer can be constructed by gene engineering method, to be applied in diagnosis or research on drugs for treating inflammatory diseases. The gene and the polypeptide establish foundation for further research on related drugs for treating rheumatoid arthritis.

Description

Technical field [0001] The invention relates to the fields of biotechnology and medicine, especially the gene and polypeptide of alpaca heavy chain VHH antibody against cyclophilin A (cyclophilin A, CypA) antigen, and the application of the gene and polypeptide. Background technique [0002] The global incidence of rheumatoid arthritis (RA) is 1%, and there are more than 5 million RA patients in my country. The disability rate of RA patients is high. Within the first 2 years of onset, about 75% of patients have joint damage, and 80% of patients suffer from joint deformation and disability after 20 years. Some diseases progress rapidly and even lead to death. Therefore, the treatment of RA has always been the focus of medical attention. In recent years, RA treatment drugs have been continuously developed, especially the application of a series of new biological agents has significantly improved synovial inflammation in RA patients and slowed down joint damage. However, the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/13C07K16/18A61K48/00A61K39/395A61P29/00A61P19/02
Inventor 陈志南汪莉朱平马小魁李郁贾俊峰杨向民
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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