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Whole molecular IgG antibody of whole human-derived anti-CD47 and application thereof

A fully human, DNA molecule technology, applied in applications, antibodies, anti-animal/human immunoglobulins, etc., can solve the problems of cumbersome preparation and low expression, and achieve the promotion of phagocytosis, good affinity, and high specificity. Effect

Active Publication Date: 2016-11-16
秦皇岛未名健长幸医疗健康科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, most domestic anti-CD47 monoclonal antibody preparation technologies are mouse-derived, or expressed after genetic engineering, but the expression level is not high or the preparation is cumbersome

Method used

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  • Whole molecular IgG antibody of whole human-derived anti-CD47 and application thereof
  • Whole molecular IgG antibody of whole human-derived anti-CD47 and application thereof
  • Whole molecular IgG antibody of whole human-derived anti-CD47 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation of fully human whole molecule IgG antibody against CD47

[0031] 1) Using the CD47 antigen in the human Fab phage library to undergo six rounds of "adsorption-elution-amplification" enrichment screening to obtain the anti-CD47 Fab antibody, and then obtain the variable region sequence of the Fab antibody by PCR amplification and sequencing.

[0032] 2) Design primers according to the obtained antibody heavy and light chain variable region sequences.

[0033] 3) Amplify the heavy chain and light chain of the anti-CD47 antibody.

[0034] Using the human Fab template prepared above, use the above-mentioned upstream and downstream primers for the heavy chain and light chain to amplify the heavy chain and light chain genes of the whole molecule human antibody, respectively.

[0035] (1)PCR

[0036] The reaction system is as follows:

[0037]

[0038] The reaction conditions are as follows:

[0039]

[0040] (2) 2% agarose gel electrophoresis, ...

Embodiment 2

[0063] Example 2 Identification of Functional Activity of Anti-CD47 Antibody

[0064] 1) ELISA

[0065] Coat ELISA 96-well plate with coating solution (0.1M carbonate buffer, pH9.6) CD47 protein to 2 μg / mL, add 100 μL to each well, overnight at 4°C; PBST (PBS containing 0.5% Tween20) 5% skimmed milk -Wash buffer to block, incubate at 37°C for 2h; after washing with PBST 5 times, add 100μL PA21 antibody (2μg / mL initial concentration, 14 concentration gradient dilutions) to each well for 2h at 37°C; goat antibody diluted 1:4000 Add 100 μL / well of human secondary antibody to the well, incubate at 37°C for 1 hour; 100 μL / well of peroxidase substrate chromogenic solution, stop the reaction with 2M sulfuric acid after 10 minutes at room temperature, and use dual-wavelength 450nm / 690nm.

[0066] The result is as figure 2 shown by figure 2 It can be seen that the anti-CD47 antibody can react with the CD47 protein.

[0067] 2) Western blot

[0068] With the rat myeloma cell YB...

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Abstract

The invention discloses a whole molecule IgG antibody of a whole human-derived anti-CD47. The whole molecular IgG antibody contains a light chain variable region and a heavy chain variable region; the amino acid sequence of the light chain variable region is as shown in a SEQIDNO.3, or a conservative variant obtained by addition, deletion, replacement and modification on one or more amino acids of the sequence; the amino acid sequence of the heavy chain variable region is as shown in a SEQIDNO.4, or a conservative variant obtained by addition, deletion, replacement and modification on one or more amino acids of the sequence. The invention also discloses a DNA molecule, an expression vector, a host cell and the application of the whole molecular IgG antibody.

Description

technical field [0001] The invention relates to the technical field of biological immunity, in particular to a fully human anti-CD47 full-molecule IgG antibody, and also relates to the DNA molecule, expression vector, host cell and application of the full-molecule IgG antibody. Background technique [0002] Macrophages remove pathogens and damaged or senescent cells from the blood through phagocytosis. CD47 on the cell surface can interact with signal regulatory protein α (SIRPα) on macrophages, thereby inhibiting the phagocytosis of normal cells. CD47 is a widely expressed transmembrane glycoprotein, containing an immunoprotein-like domain and 5 transmembrane regions, which constitute the ligand of SIRPα, and bind SIRPα through the variable region-like domain at the NH2 terminal. SIRPα is mainly expressed in myeloid cells, including macrophages, granulocytes, myeloid dendritic cells, mast cells, and their precursors, including hematopoietic stem cells. SIRPα can inhibit h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C07K16/30C12N15/13G01N33/574A61K39/395A61P35/00
CPCA61K2039/505C07K16/18C07K16/30C07K2317/24C07K2317/52C07K2317/56C07K2317/565C07K2317/92
Inventor 冯振卿朱进许国贞刘振云熊四平唐奇
Owner 秦皇岛未名健长幸医疗健康科技有限公司
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