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Mica extracellular region mutant and its screening method, scfv-mica fusion antibody and its preparation method and application

A fusion antibody and scfv-mica technology, which is applied in the field of protein engineering, can solve the problems of limiting the strength of immune surveillance, anti-tumor efficacy, and limited affinity, and achieves low operational difficulty and condition requirements, significant tumor growth, and mature technology Effect

Active Publication Date: 2022-04-12
CHANGSHA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the limited affinity of MICA to the receptor NKG2D limits the strength of this pathway's immune surveillance and antitumor efficacy

Method used

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  • Mica extracellular region mutant and its screening method, scfv-mica fusion antibody and its preparation method and application
  • Mica extracellular region mutant and its screening method, scfv-mica fusion antibody and its preparation method and application
  • Mica extracellular region mutant and its screening method, scfv-mica fusion antibody and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] A MICA extracellular region mutant of the present invention is constructed by the following method:

[0061] (1) Construction of mutant MICA phage display library:

[0062] 1.1. Download the extracellular region sequence of MICA from genbank, GeneID: 100507436, Protein: CAA77031.1.

[0063] 1.2. Using the sequence in step 1.1 as a template, amplify the MICA extracellular region gene fragment by mismatch PCR and introduce random mutations to obtain mutations, and construct a mutant MICA gene library; the specific steps are:

[0064] Design primers:

[0065] MICA-F (SEQ ID NO. 2): GAACCTCATTCTCTGC;

[0066] MICA-R (SEQ ID NO. 3): ACCAGATTTCAGATAGC.

[0067] PCR reaction system:

[0068] Mg in the reaction system 2+ The final concentration is 5mmol / L.

[0069] PCR reaction program: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 52°C renaturation for 30 s, 72°C extension for 60 s, 30 cycles, and finally 72°C extension for 10 min.

[0070] 1.3. Insert ...

Embodiment 2

[0118] A scFv-MICA fusion antibody of the present invention is constructed by the following method:

[0119] (1) Download the sequence of cetuximab from NCBI, and obtain the sequences of the variable regions of the light and heavy chains, the heavy chain is 5T1M_D, and the light chain is 5T1M_C. The light and heavy variable region sequences were linked with a flexible peptide GGGGS to construct a single-chain antibody scFv.

[0120] (2) The scFv is fused with the MICA extracellular region mutant sequence of Example 1, and linked with a G4S flexible peptide to construct a recombinant gene of the scFv-mMICA fusion antibody molecule.

[0121] (3) A His tag was introduced into the recombinant gene, and pPICZα was inserted to construct a recombinant vector.

[0122] (5) The recombinant vector was transfected into Pichia pastoris X-33, screened with bleomycin, identified by Western blot, expanded and fermented (the above method is the same as in Example 1) to obtain the fusion anti...

Embodiment 3

[0135]The application of the scFv-MICA fusion antibody of Example 2 in anti-tumor in vitro uses the CytoTox 96 non-radioactive cytotoxicity assay based on the colorimetric method. CytoTox 96 can quantitatively measure the content of lactate dehydrogenase (LDH). Lactate dehydrogenase is a level-stable cytosolic enzyme that is released into the medium supernatant when cells are lysed. LDH can convert tetrazolium salt (INT) into red formazan. The amount of formazan generated is directly proportional to the number of lysed cells, and the number of lysed cells is deduced by detecting the concentration of formazan. The effector cells used in the experiment were PBMC cells or FcγRIIIa-modified NK92 cells cultured in vitro, and the target cells were HT-29 and A431. FcγRIIIa-modified NK92 cells, HT-29 and A431 cells in logarithmic growth phase were collected. Its specific application method is:

[0136] (1) Setting of the detection board:

[0137] 1.1. Spontaneous release of LDH by...

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Abstract

The invention discloses a MICA extracellular region mutant, a screening method thereof, a scFv-MICA fusion antibody, a preparation method and application thereof. Mutations in the 24th, 33rd, 69th, 112th, and 126th positions of MICA extracellular region mutants. Screening methods: construction of mutant MICA phage library, expression of recombinant protein NKG2D-Fc, phage screening, etc. The scFv‑MICA fusion antibody consists of a scFv linked to a MICA mutant with a flexible peptide. Preparation method: construction of recombinant gene of fusion antibody; construction of recombinant vector; transfection of competent cells, expression and purification, etc. The extracellular region mutant of MICA of the present invention can efficiently induce the cytotoxic effect of NK cells mediated by NKG2D, and the fusion antibody constructed with scFv can effectively target tumor cells by using the tumor-specific antibody part, and at the same time, the high-affinity mutant MICA part can be Mediates NK cells and other effective killing of target cells.

Description

technical field [0001] The invention relates to the technical field of protein engineering, in particular to a MICA extracellular region mutant and its screening method, scFv-MICA fusion antibody and its preparation method and application. Background technique [0002] MICA-NKG2D-mediated immune surveillance: Natural killer cells (NK cells) are an important part of the innate immune system, have non-specific immune killing functions, and are the body's first line of defense against infection, anti-tumor and removal of target cells . On the surface of NK cells, NKG2D (natural killer cell receptor G2D) is an important activating receptor, which can effectively activate NK under the stimulation of its ligand MHC-I (major histocompatibility complex-I) related antigen molecule MICA / MICB Cells, play a non-specific immune killing effect. [0003] Insufficiency and solution of MICA-NKG2D-mediated immune surveillance: MICA is concentratedly expressed in gastrointestinal epithelial ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/74C12N15/12C12N15/81C07K19/00A61K38/17A61K47/68A61P35/00
CPCC07K14/70539C12N15/815C07K16/2833A61K38/1774A61K47/6811A61K47/6851A61P35/00C07K2317/622
Inventor 陈治国曾志红袁依俊范紫凌徐文倩
Owner CHANGSHA UNIVERSITY
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