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C5aR antibody, preparation method and application thereof

A technology of antibody and sequence, applied in the field of C5aR antibody and its preparation

Active Publication Date: 2017-07-18
SHANGHAI YUNYI HEALTHCARE MANAGEMENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still no new antagonist drugs targeting C5aR and its ligands on the market.

Method used

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  • C5aR antibody, preparation method and application thereof
  • C5aR antibody, preparation method and application thereof
  • C5aR antibody, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Preparation of C5aR antibody

[0106] (1), preparation of immunogen A

[0107] C5aR is a seven-transmembrane protein, and its extracellular domain includes an N-terminal and three extracellular loops. The following five polypeptides were designed for the N-terminal and three extracellular loops of the C5aR protein respectively, and the specific sequences are shown in Table 3. The peptides shown in Table 3 were synthesized by Jill Biochemical Shanghai Co., Ltd. Immunogen A is obtained by coupling these polypeptides with keyhole limpet hemocyanin (KLH). For the coupling method, see "Establishment and Application of PRRS Antibody Indirect ELISA Detection Method" [J]. China Veterinary Science and Technology, 2005 (6).

[0108] Table 3 The polypeptide sequence of immunogen A

[0109]

[0110]

[0111] (2) Preparation of immunogen B

[0112] The stable cell line used in immunogen B was infected by lentivirus. The nucleotide sequence encoding the full-length amino ...

Embodiment 2

[0128] Production and purification of lead antibodies

[0129] The antibody concentration produced by hybridoma cells is low, only about 1-10 μg / ml, and the concentration varies greatly. Moreover, various proteins produced by cell culture in the culture medium and fetal bovine serum components contained in the culture medium have varying degrees of interference with many biological activity analysis methods, so small-scale (1-5 mg) antibody production and purification are required.

[0130] The hybridoma cells obtained in Example 1 were inoculated into T-75 cell culture flasks and acclimatized and passaged for 3 generations with a production medium (Hybridoma serum free medium, purchased from Invitrogen). When the growth state is good, inoculate the cell culture spinner bottle. Add 500 ml of production medium to each 2-liter culture spinner bottle, and inoculate the cell density at 1.0×10 5 / ml. Close the bottle cap tightly, and place the spinner bottle on a spinner machine...

Embodiment 3

[0137] Assay of lead antibody

[0138] A. Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding of antibody to C5aR protein. The purified C5aR antibody obtained in Example 2 was cross-reacted with the five polypeptides of the extracellular domain of C5aR, that is, the purified immunogen A obtained in Example 1, respectively.

[0139] The five polypeptides of the purified immunogen A obtained in Example 1 were respectively diluted with PBS to a final concentration of 1.0 μg / mL, and then added to a 96-well ELISA plate at 100 μl per well. Seal it with a plastic film and incubate overnight at 4°C. The next day, wash the plate twice with plate washing solution [PBS containing 0.01% (v / v) Tween20], add blocking solution [containing 0.01% (v / v) Tween20 and 1 % (v / v) BSA in PBS] for blocking at room temperature for 2 hours. The blocking solution was discarded, and 100 μl of the purified C5aR antibody obtained in Example 2 was added to each well. After incubation ...

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Abstract

The invention discloses a C5aR antibody, a preparation method and an application thereof. The C5aR antibody comprises one or more of a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3 in a heavy-chain variable region, and one or more of a light chain CDR1, a light chain CDR2 and a light chain CDR3 in a light-chain variable region. The C5aR antibody has the advantages that the affinity for a human-derived C5aR protein is high, the combination of the C5aR protein and C5a can be effectively prevented, so that corresponding signal channels are descended or cut off, and directional migration of C5a of in-vitro neutrophil is stopped; and the chemotactic effect of the neutrophil can be obviously inhibited. Therefore, the C5a antibody can be applied to preparation of medicines for treating inflammations, vascular diseases or nervous system diseases and the like.

Description

technical field [0001] The present invention relates to the field of antibodies, in particular to a C5aR antibody and its preparation method and application. Background technique [0002] Complement system is composed of more than 30 kinds of plasma proteins and membrane proteins, widely exists in blood, interstitial fluid and cell surface, and is a protein reaction system with precise regulation mechanism. The main physiological function of complement is to promote the phagocytosis of phagocytes and dissolve target cells, so it is an important part of the body's immune defense mechanism. [0003] The ligand C5a of C5aR is an activation product of the complement system and an important mediator and chemokine of the inflammatory response. The combination of C5aR and ligand C5a induces an inflammatory response and affects the coagulation and fibrinolytic system, resulting in damage to normal tissue cells, which participates in the pathological process of various diseases. C5...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63C12N1/21C12N5/10G01N33/53
CPCC07K16/28C12N5/10C12N15/63C07K16/2896C07K2317/14C07K2317/30C07K2317/70C07K2317/76C07K2317/92G01N2333/70596G01N2333/4716C07K2317/565G01N33/6893
Inventor 杨翠青段清徐丽娜顾红专刘礼乐
Owner SHANGHAI YUNYI HEALTHCARE MANAGEMENT
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