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Human PD-L1 protein Y123 site phosphorylation antibody as well as preparation method and application thereof

A technology of PD-L1 and phosphorylation, applied in chemical instruments and methods, anti-animal/human immunoglobulins, antibodies, etc. drug problems

Active Publication Date: 2017-10-27
广州华科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the research on PD-1 / PD-L1 is relatively concentrated, but the development of new targets is lacking, and the treatment mode of "immunotherapy" is not standardized enough
Although PD-1 inhibitors are fundamentally different from targeted drugs (for cancer cells), the problem of drug resistance still exists, and further research and development of new targets are necessary

Method used

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  • Human PD-L1 protein Y123 site phosphorylation antibody as well as preparation method and application thereof
  • Human PD-L1 protein Y123 site phosphorylation antibody as well as preparation method and application thereof
  • Human PD-L1 protein Y123 site phosphorylation antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Determine the phosphorylation site of human PD-L1 protein

[0032] First, the bioinformatics software NetPhorest 2.0 and NetPhos 2.0 were used to screen possible phosphorylation sites in the human PD-L1 protein, and then the mass spectrometry literature and protein phosphorylation site database PhosohoSitePlus were searched to determine Y 123The site is a phosphorylation site in the amino acid sequence of human PD-L1; at the same time, the antigenicity and hydrophilicity of the amino acid sequence of human PD-L1 are calculated using the software CLC ProteinWorkbench 5, and the specific phosphorylation site of human PD-L1 protein is finally determined The point is the 123rd amino acid - Tyrosine Y 123 .

[0033] Human PD-L1 protein has multiple phosphorylation sites (such as Y 28 site), because the subsequent antigenicity and hydrophilicity do not meet the test requirements, it is not suitable as a phosphorylation site.

Embodiment 2

[0034] Embodiment 2 synthetic hapten synthetic peptide

[0035] According to the design of hapten synthetic peptides, in Y 123 A phosphorylation group is added to the site, and HPLC (high performance liquid chromatography) separation and purification are performed to obtain a hapten synthetic peptide with a purity of more than 95%. Correspondingly, a piece of Y is synthesized 123 The site non-phosphorylated synthetic peptide is coupled with bovine serum albumin through N-terminal cysteine ​​and used as filler for affinity separation chromatography. The purity after separation and purification by HPLC is over 90%.

Embodiment 3

[0036] Embodiment 3 prepares antiserum

[0037] 3.1 Preparation of negative serum

[0038] 3 mL of blood was collected from the ear vein of New Zealand white rabbits (2-3 kg, female, robust) used for injection into blood collection tubes, and the bleeding was stopped by pressing with cotton balls. Leave the blood at room temperature for about 1 hour, wait for the blood to coagulate to form blood clots, place it at 4°C for 2 hours to precipitate the serum, centrifuge at 2500g for 10 minutes, absorb the supernatant, label it as negative control serum, aliquot and store at -20°C for testing.

[0039] 3.2 Screening before immunization - Western blot (Western blot):

[0040] BCA (Bicinchoninic acid) Protein Concentration Detection Kit Determination of Dissolved Phosphoantigen (pY 123 -BSA) concentration and non-phosphorylated antigen (Y 123 - BSA) concentration, respectively 0.845 mg / mL and 1.1 mg / mL (correlation coefficient R = 0.991). Will pY 123 -BSA and Y 123 -BSA for pro...

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Abstract

The invention discloses a human PD-L1 protein Y123 site phosphorylation antibody as well as a preparation method and application thereof, and belongs to the technical field of biomedicine of antibody preparation. According to the preparation method of the human PD-L1 protein Y123 site phosphorylation antibody, semiantigen synthetic peptide is coupled with carrier protein through cysteine C to obtain antigen; the antigen in the step (1) is used for performing immunization on animals; antiserum is collected; the antiserum in the step (2) is purified and selected; the PD-L1 protein Y123 site phosphorylation antibody is obtained; the amino acid sequence of the semiantigen synthetic peptide is CSYGGADYKRITVK; a phosphate group is added to the tyrosine Y site of the semiantigen synthetic peptide. The human PD-L1 protein Y123 site phosphorylation antibody provided by the invention can be applied to the malignant tumor diagnosis, treatment and prognosis judgment.

Description

technical field [0001] The invention belongs to the field of biomedical technology for antibody preparation, in particular to a human PD-L1 protein Y 123 Site phosphorylated antibody and its preparation method and application. Background technique [0002] According to the 2016 global cancer statistics, there were approximately 15 million new cancer cases and 8.2 million cancer deaths worldwide throughout the year; among them, 57% of cancer patients and 65% of cancer deaths came from developing countries. The occurrence and development of tumors is a very complicated process, and T cells play a vital role in the body's anti-tumor immune system. In order to evade the surveillance of the body's immune system, tumor cells upregulate the expression of programmed cell death ligand 1 (programmed cell death lagand 1, PD-L1, also known as B7-H1) in the tumor microenvironment through various mechanisms, PD- L1 binds to a negative immune checkpoint on the surface of T cells, the pro...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K16/28C07K16/06G01N33/574A61K39/395A61P35/00
CPCA61K39/00C07K7/08C07K16/065C07K16/2827C07K2317/21C07K2317/30C07K2317/33G01N33/57488
Inventor 张灏
Owner 广州华科生物科技有限公司
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