Fusion protein of anti-C3d targeted single-chain antibody and CD59, and application of fusion protein

A fusion protein and single-chain antibody technology, applied in the field of fusion proteins, can solve problems affecting the targeting effect of anti-C3d antibodies, increase protein molecular weight, etc., and achieve excellent anti-adhesion/anti-inflammatory targeting inhibition effect, vasculitis improvement, high The effect of suppressing efficiency

Active Publication Date: 2020-03-27
BEIJING COMPLEMENT THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the single-chain antibody of the anti-C3d antibody in the prior art is still difficult to meet this technical requirement. The fusion of the anti-C3d antibody and different inhibitors will increase the molecular weight of the protein, which will change the spatial structure of the protein after reconstruction, thereby affecting Targeting effect of anti-C3d antibodies

Method used

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  • Fusion protein of anti-C3d targeted single-chain antibody and CD59, and application of fusion protein
  • Fusion protein of anti-C3d targeted single-chain antibody and CD59, and application of fusion protein
  • Fusion protein of anti-C3d targeted single-chain antibody and CD59, and application of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, preparation of anti-C3d single chain antibody

[0026] Use the following method to screen anti-C3d single-chain antibody, and the specific method includes the following steps:

[0027] 1.1 Preparation of cDNA

[0028] 20 ml of peripheral blood from 50 healthy individuals were collected, and mononuclear cells were separated with lymphocyte separation medium (Tianjin Blood Research Institute). Total RNA was extracted from isolated human peripheral blood lymphocytes with Trizol reagent (Invitrogen), and then mixed in the same ratio. The cDNA was reverse transcribed using a cDNA reverse transcription kit (Takara Company). The above steps were carried out according to the instructions provided by the manufacturer.

[0029] 1.2 Amplification of antibody light chain and heavy chain variable region genes: Using PCR method, using the cDNA synthesized by reverse transcription as a template, add primers for amplifying the light chain and heavy chain variable regio...

Embodiment 2

[0050] Example 2 Preparation of anti-C3d single-chain antibody-CD59 (ScFv-CD59) fusion protein

[0051] 1. Materials The expression vector was pEE14.1 (Lonza biologicals); CHO cells were used for protein expression, and the culture medium was DMEM containing 10% fetal bovine serum, which was purchased from Invitrogen. Mouse anti-CD59 mAb 1H4 and 1A10, mouse anti-human CR2 mAb 171, anti-goat erythrocyte IgM and all secondary antibodies were purchased from Sigma.

[0052] 2 methods

[0053] 2.1 The preparation of rabbit anti-CHO cell membrane and human CD59 antiserum was described in Harlow, E., and Lane, D. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, New York, USA. 1988: 726. method to obtain.

[0054] 2.2 Construction of expression recombinant and protein expression The cDNA structural gene was formed by linking the sequence encoding the anti-C3d single-chain antibody and the sequence encoding the extracellular region of CD59. The com...

Embodiment 3

[0074] Example 3. Kinetic analysis of interaction between ScFv-CD59 and C3 ligand

[0075] Kinetic analysis of the interaction of ScFv–CD59 with biotin-labeled C3dg (C3dg-biotin) was performed using a surface plasmon resonance (SPR) detection system (BIAcore 3000 instrument). Human C3dg-biotin (Guthridge, J.M., et al. Structural studies solution of the recombinant N-terminal pair of short consensus / complement repeat domains of complement receptor type 2 (CR2 / CD21) and interactions with its ligand C3dg.Biochemistry.2001,40(20):5931–5941.) were injected onto the BIAcore streptavidin sensor chip at a speed of 2μL / min for 20min, and the buffer was 0.5× PBS (pH7.4) (containing 0.5g / L Tween20). SPR signals acquired from captured C3dg yielded BIAcore response units (range 250 to 500). The group without fusion protein was used as the control. After washing with 0.5×PBST (0.5g / LTween20) at a flow rate of 25μL / min at 25°C, the affinity of ScFv-CD59 was evaluated by detecting the con...

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Abstract

The invention discloses a fusion protein of a single-chain antibody of a human anti-complement C3d molecule and a complement inhibitor CD59. The antibody has excellent antigen binding activity. Biological distribution experiments prove that the fusion protein provided by the invention can be rapidly and highly aggregated at an arthritis part after entering a rheumatoid arthritis mouse model, and has an excellent anti-adhesion/anti-inflammation targeting inhibition effect. In treatment of MRL/lpr lupus erythematosus mice, the fusion protein provided by the invention can obviously improve the survival rate of the mice, and symptoms of proteinuria, glomerular score, interstitial inflammation, vasculitis, crescent/necrosis and the like of a treatment group are obviously improved, so that the fusion protein provided by the invention has an excellent application prospect in preparation of medicaments for treating autoimmune diseases.

Description

technical field [0001] The invention provides a fusion protein and belongs to the technical field of polypeptides. Background technique [0002] The complement system is composed of more than 30 kinds of soluble protein molecules and is a part of the natural immune system. Its components include more than 30 kinds of molecules such as complement intrinsic components, various regulatory factors and complement receptors. The complement system can be activated through three relatively independent and interrelated pathways, thereby exerting various biological effects such as opsonizing phagocytosis, lysing cells, mediating inflammation, immune regulation, and clearing immune complexes, including enhancing phagocytosis, enhancing phagocytosis Chemotaxis of cells; increase of vascular permeability; neutralization of viruses; cell lysis; regulation of immune response, etc. Cell or tissue destruction can also be caused indirectly by complement activation and its deposition on targe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K39/395A61P29/00A61P19/02A61P37/02A61K38/17
CPCA61K38/177A61K39/39533A61P19/02A61P29/00A61P37/02C07K14/70596C07K16/18C07K2317/24C07K2317/565C07K2319/00C12N15/85A61K2300/00
Inventor 唐晓敏杜兰英
Owner BEIJING COMPLEMENT THERAPEUTICS LTD
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