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Design and application of CD24 antibody fusion protein

A technology of fusion protein and CD24, applied in the field of body specific binding

Active Publication Date: 2016-06-29
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are few reports on fusion proteins related to NKG2D ligand MICA molecules

Method used

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  • Design and application of CD24 antibody fusion protein
  • Design and application of CD24 antibody fusion protein
  • Design and application of CD24 antibody fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1CD24

[0035] Example 1 Construction of CD24 Antibody Fusion Protein rG7S-MICA

[0036] Firstly, CHO-biased codon optimization was performed on the genes of CD24 single-chain antibody rG7S and the extracellular region 1-3 of human MICA. Further, using the optimized two genes as templates, overlapPCR technology was used to pass the flexible peptide G 4 S was used for gene splicing and enzyme cutting sites at both ends were added, the PCR products were detected by 1% agarose gel electrophoresis, and the target gene fragment was recovered by the agarose gel recovery kit. The target gene and pMH3 / pCApuro were digested separately, and the target gene and plasmid fragments were recovered after digestion. T4 ligase was ligated overnight at 16°C to obtain two recombinant plasmids (rG7S-MICA-pMH3, rG7S-MICA-PCApuro). CaCl 2 The ligation product was transformed into Escherichia coli DH5α for amplification and storage.

Embodiment 2CD24

[0037] Example 2 Expression and purification of CD24 antibody fusion protein rG7S-MICA

[0038] First, the two recombinant plasmids rG7S-MICA-pMH3 and rG7S-MICA-PCApuro were introduced into CHO-s cells by electric shock method (in a 0.4cm electric shock cup, 160V, 15ms) in equal proportions, and two rounds of pressurized selection (DotBlot Dot blot semi-quantitative screening) to obtain cell lines stably expressing rG7S-MICA. The screened high-expression cell lines were expanded step by step, the cell culture fluid was collected, and the samples were filtered through a 0.22 μm filter membrane, followed by nickel ion affinity chromatography purification, and finally a large amount of the target protein was obtained. 12% SDS-PAGE protein electrophoresis and Western blot for preliminary verification of the target protein.

Embodiment 3

[0039] Example 3 Affinity analysis of rG7S-MICA by micro-thermophoresis technique

[0040] In this experiment, MonolithNT.115 was used as an MST-dependent detection instrument to analyze the interaction between the antibody fusion protein rG7S-MICA and CD24 / NKG2D: A. Using MonolithNT TM The protein labeling kit is used for fluorescent molecular labeling of rG7S-MICA. A constant concentration of rG7S-MICA was mixed with serially diluted CD24 naked peptide or NKG2D molecules, each sample was sucked by a standard capillary, and the intermolecular affinity constant was determined by detecting the change in the thermophoretic efficiency of protein molecules after rG7S-MICA and CD24 / NKG2D combined.

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Abstract

The invention belongs to the technical field of genetic engineering antibodies, and particularly relates to a preparation method and application of CD24 antibody fusion protein. The preparation method includes that genetic engineering technology is utilized to connect CD24 single-strand antibody rG7S with MICA extracellular 1-3 zones through G4S flexible peptide, and a mammal eukaryotic expression system stably expresses rG7S-MICA; the CD24 antibody fusion protein rG7S-MICA can be combined with CD24 molecules on the surface of tumor cells and an activated receptor NKG2D on the surface of NK cells at the same time, and can remodel immunological surveillance effect, on CD24+tumor cells, of NK cells induced through an NKG2D path to finally achieve the objective of activating a body autoimmune system to effectively kill the CD24+tumor cells.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a new CD24 antibody fusion protein rG7S-MICA that can specifically bind to the leukocyte differentiation antigen CD24 (clusterofdifferentiation24) and NKG2D receptors at the same time, and uses the targeting of rG7S to specifically bind MICA It is displayed on the surface of tumor cells, avoiding the escape of tumor immunity caused by the down-regulation and shedding of MICA molecule expression on the cell surface. In vitro and in vivo assays, CD24 + MICA on the surface of liver cancer cells Huh-7 effectively recruits and activates NK cells through the MICA-NKG2D signaling pathway, induces them to release perforin-granzyme, secretes IFN-γ, TNF-α and other cytokines, effectively kills tumor cells and inhibits its growing. The design of rG7S-MICA opens up a new strategy for tumor immunotherapy and is a specific remodeling NK cell response to CD24 + A bifunctional antibody ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/00A61K47/48A61P35/00
Inventor 张娟王旻王彤孙福谋
Owner CHINA PHARM UNIV
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