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Cell surface macromolecule quantitative display system as well as preparation method and application thereof

A display system and cell surface technology, which is applied in the field of cell surface macromolecule quantitative display system and its preparation, can solve the problems of not being able to distinguish the difference of antibody-antigen affinity well

Pending Publication Date: 2022-05-06
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported synNotch synthesis system as an antibody expression system does not distinguish antibody-antigen affinity differences well

Method used

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  • Cell surface macromolecule quantitative display system as well as preparation method and application thereof
  • Cell surface macromolecule quantitative display system as well as preparation method and application thereof
  • Cell surface macromolecule quantitative display system as well as preparation method and application thereof

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preparation example Construction

[0066] The present invention also provides a preparation method of a cell surface macromolecule quantitative display system, the preparation method comprising the following steps:

[0067] 1) Cloning the receptor of interest onto the cell membrane of the receptor-expressing cell;

[0068] 2) cloning the ligand of interest onto the ligand quantitatively presenting cell;

[0069] The methods of cloning the target receptor into receptor expressing cells and cloning the target ligand into the ligand quantitatively presenting cells are molecular biology methods well known to those skilled in the art.

[0070] The receptor expressing cells carry a fluorescent reporter group.

[0071] The ligands and receptors mentioned in the present invention can be antigens and antibodies.

[0072] The present invention also provides the use of the cell surface macromolecule quantitative display system in ligand-receptor affinity detection and / or batch acquisition of high-affinity antibodies.

...

Embodiment 1

[0101] Example 1 quantitatively demonstrates the construction and activation of the desired stable transfection cell line

[0102] Construction: express the GFP-mem fusion protein on the surface of the ligand-presenting cell (Ag-mem APC) by the following method, and express the GFP-m-LC fusion protein on the surface of the ligand quantitative presentation cell (Ag-m-LC APC), and The fusion GFP and mCherry nanobodies were expressed on the surface of K562 cells (anti-GFP: KD=0.31 μM, anti-mcherry: KD=0.18 nM, the antibodies were derived from Fridy, P.C. et al. A robust pipeline for rapid production of versatile nanobody repertoires. Nat.Methods 11,1253-1260, doi:10.1038 / nmeth.3170 (2014) synNotch synthetic receptor, this K562 cell is called receptor expressing cell or antibody expressing cell (ABC) ( figure 1 A, 1B). The method is as follows: the retrovirus corresponding to the expression vector is obtained by transient transfection with liposomes in 293T by a conventional meth...

Embodiment 2

[0105] Example 2 Mammalian Cell Surface Antibody Display System Quantitative Detection of Ligand-Receptor Binding Ability

[0106] To examine the effect of intermolecular affinity on synNotch synthetic receptor activation, GFP-APCs were co-cultured with two anti-GFP synNotch ABC cell lines at a 2:1:1 ratio for 48 h. Although the affinities of the two anti-GFP Nanobodies differed by two orders of magnitude ("Hi": KD = 3.5 nM; "Lo": KD = 600 nM), similar affinities were observed in both anti-GFP synNotch cells. The proportion of BFP positive cells ( figure 1 E). In addition, mix APC / ABC at a ratio of 1:3 or 1:9 to reduce the antigen content by reducing the number of APC cells in the system, so that ABC of high-affinity antibodies can gain a competitive advantage under extreme conditions.

[0107] The results showed that anti-GFP at 1:9 Hi The proportion of BFP-positive cells in SynNotch ABC was slightly increased ( figure 1 E). Thus, molecular interactions with affinities ...

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Abstract

The invention relates to the technical field of biology, in particular to a cell surface macromolecule quantitative display system and a preparation method and application thereof, and the cell surface macromolecule quantitative display system comprises ligand quantitative presenting cells and receptor expression cells. Wherein a phase change peptide fragment is fused on a transmembrane protein of the ligand quantitative presenting cell, the receptor expression cell is a synNotch synthetic receptor expression cell, and an extracellular region of the synNotch synthetic receptor is fused with a region which can be specifically recognized and combined with a ligand on the surface of the ligand presenting cell. When the ligand quantitative presenting cell and the receptor expression cell are co-cultured, the receptor of the receptor expression cell can be specifically and quantitatively activated. The cell surface macromolecule quantitative display system can be applied to ligand-receptor (including antigen-antibody) affinity detection and / or batch acquisition of high-affinity antibodies, the batch of high-affinity antibodies can be rapidly obtained, and subsequent tedious verification work of technologies such as phage display is avoided.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a system for quantitatively displaying macromolecules on the cell surface, a preparation method and application thereof. Background technique [0002] How mammalian cells perceive and distinguish the magnitude of affinity between cell surface receptors and their corresponding ligands when cells are in contact with each other, and convert them into specific quantitative biological events is still a scientific mystery. For example, the mechanism by which lymphatic B cells expressing high-affinity antibodies (i.e., B-cell receptors) compete with low-affinity B cells for antigen molecules displayed on the surface of antigen-presenting cells remains unclear. [0003] Antibody affinity maturation is a hallmark of the immune response of the adaptive immune system, during which high-affinity antibodies against a specific antigen are produced. Antibody affinity maturation in germinal centers...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K16/18C12N15/62C12N5/10G01N33/53
CPCC07K14/71C07K16/18G01N33/53C07K2319/00C12N15/62C07K19/00C12N5/10C12N15/00G01N33/68
Inventor 孟飞龙刘浏蔡燕妮
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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