MICA extracellular region mutant, screening method thereof, scFv-MICA fusion antibody and preparation method and application of scFv-MICA fusion antibody

A screening method and antibody fusion technology, applied in the field of protein engineering, can solve the problems of limiting the strength of immune surveillance and anti-tumor efficacy, limited affinity, etc., and achieve the effect of low operational difficulty and condition requirements, significant tumor growth, and mature technology

Active Publication Date: 2020-07-31
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, screening method thereof, scFv-MICA fusion antibody and preparation method and application of scFv-MICA fusion antibody
  • MICA extracellular region mutant, screening method thereof, scFv-MICA fusion antibody and preparation method and application of scFv-MICA fusion antibody
  • MICA extracellular region mutant, screening method thereof, scFv-MICA fusion antibody and preparation method and application of scFv-MICA fusion antibody

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]

[0069] Mg in the reaction system 2+ The final concentration is 5mmol / L.

[0070] 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.

[0071] 1....

Embodiment 2

[0119] A scFv-MICA fusion antibody of the present invention is constructed by the following method:

[0120] (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.

[0121] (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.

[0122] (3) A His tag was introduced into the recombinant gene, and pPICZα was inserted to construct a recombinant vector.

[0123] (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

[0136]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:

[0137] (1) Setting of the detection board:

[0138] 1.1. Spontaneous release of LDH by...

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Abstract

The invention discloses an MICA extracellular region mutant, a screening method thereof, an scFv-MICA fusion antibody, and a preparation method and application of the scFv-MICA fusion antibody. The MICA extracellular region mutant is mutated at the 24th site, the 33th site, the 69th site, the 112th site and the 126th site. The screening method comprises the following steps: constructing a mutant MICA bacteriophage library, expressing recombinant protein NKG2D-Fc, screening bacteriophage and the like. The scFv-MICA fusion antibody is formed by linking scFv and an MICA mutant through a flexiblepeptide. The preparation method comprises the following steps: constructing a recombinant gene of a fusion antibody, constructing a recombinant vector, transfecting competent cells, performing expressing and purifying, and the like. The MICA extracellular region mutant disclosed by the invention can be used for efficiently inducing the cytotoxic effect of NKG2D mediated NK cells; the fusion antibody constructed by the MICA extracellular region mutant and scFv can utilize a tumor specific antibody part to efficiently target tumor cells, and meanwhile, the high-affinity mutation MICA part can mediate NK cells and the like to effectively kill 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 Applications(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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