Complete-human-derived anti-CD45 all-molecule IgG antibody and application thereof

A fully human, DNA molecular technology, applied in the fields of application, antibody, anti-animal/human immunoglobulin, etc., can solve the problem of destroying antigen recognition ability, and achieve the effect of high specificity and good affinity

Inactive Publication Date: 2016-10-26
SINOBIOWAY CELL THERAPY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Some cells proliferate and differentiate into cy

Method used

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  • Complete-human-derived anti-CD45 all-molecule IgG antibody and application thereof
  • Complete-human-derived anti-CD45 all-molecule IgG antibody and application thereof
  • Complete-human-derived anti-CD45 all-molecule IgG antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation of fully human whole molecule IgG antibody against CD45

[0031] 1) Using the CD45 antigen in the human Fab phage library to undergo six rounds of "adsorption-elution-amplification" enrichment screening to obtain the anti-CD45 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-CD45 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-CD45 Antibody

[0064] 1) ELISA

[0065] Coat ELISA 96-well plate with coating solution (0.1M carbonate buffer, pH9.6) CD45 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-CD45 antibody can react with the CD45 protein.

[0067] 2) Western blot

[0068] Use empty bacteria that do n...

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PUM

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Abstract

The invention discloses a complete-human-derived anti-CD45 all-molecule IgG antibody which comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is disclosed as SEQ ID NO.3, or the sequence is a conserved mutant obtained by carrying out conserved mutation by addition, deletion, substitution and modification of one or more amino acids; and the amino acid sequence of the heavy chain variable region is disclosed as SEQ ID NO.4, or the sequence is a conserved mutant obtained by carrying out conserved mutation by addition, deletion, substitution and modification of one or more amino acids. The invention also discloses a DNA molecule, an expression vector, a host cell and application of the complete-human-derived anti-CD45 all-molecule IgG antibody.

Description

technical field [0001] The present invention relates to the technical field of biological immunity, in particular to a fully human anti-CD45 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] Organ, cell and tissue transplant rejection and various autoimmune diseases are currently considered to be mainly caused by T cell-mediated immune response. T cell-mediated immune responses are initially initiated by helper T cells recognizing specific antigens. These helper T cells may be legacy memory cells from previous immune responses or naive cells released from the thymus and may express either of an extremely broad range of antigen receptors. When helper T cells recognize antigen-MHC complexes on the surface of antigen-presenting cells (APCs) or macrophages, helper T cells are stimulated to release IL-2 to promote helper T cell proliferation. Proliferati...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13G01N33/68G01N33/574A61K39/395A61P37/02A61P35/02A61P37/06
CPCC07K16/289A61K2039/505C07K2317/21C07K2317/52C07K2317/56C07K2317/565G01N33/57426G01N33/6854G01N2333/70589G01N2800/24G01N2800/245
Inventor 冯振卿徐亚如许国贞刘振云唐奇朱进
Owner SINOBIOWAY CELL THERAPY CO LTD
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