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Evolution method and application for antibody

An antibody and mutant antibody technology, applied in the field of cell biology, can solve the problem of low antibody mutation efficiency, reduce site interference, improve evolution efficiency, and improve antibody affinity.

Active Publication Date: 2020-03-31
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a method for antibody evolution and its application to solve the problem of antibody mutation efficiency in the prior art low problem

Method used

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  • Evolution method and application for antibody
  • Evolution method and application for antibody
  • Evolution method and application for antibody

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Experimental program
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Effect test

Embodiment 1

[0055] Embodiment 1 The method for improving antibody affinity

[0056] 1.1 Construction of antibody surface display system

[0057] Codon optimization is performed first, and all base combinations corresponding to amino acids in the VB18 gene sequence are listed, and the most retained WRC or AGCT sequence motifs are manually selected, and handed over to Suzhou Jinweizhi Company for synthesis. The optimized nucleotide sequence of the VB18 gene is shown in SEQ ID NO:2.

[0058] The carrier used to overexpress membrane-bound antibody molecules on the surface of 293T cells is lenti dCAS-VP64_Blast, which is modified to retain the viral packaging backbone, and the lenti dCAS-VP64_Blast plasmid is digested with BsiWI and EcoRI, and the signal peptide (SEQ ID NO: 3 ), optimized VB18 gene sequence, and IgG constant region sequence (CH2-CH3) (SEQ ID NO: 4) were all synthesized in Suzhou Jinweizhi Company. The recombinant fragment was connected to the corresponding plasmid by PCR ampli...

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Abstract

The invention provides an evolution method for antibody. The evolution method for the antibody includes the following steps: 1) providing a single-base gene editing system, wherein the single-base gene editing system can form a single-base gene editing complex, the single-base gene editing complex includes a dCas9 enzyme, sgRNA, and an MS2 cytosine deaminase fusion protein, and the sgRNA targets variable region genes of an antibody to be evolved; 2) performing base mutation on the variable region genes of the antibody to be evolved by using the single-base gene editing system described in thestep 1), and expressing mutant antibodies to obtain a mutant antibody library; and 3) screening and enriching a high-affinity antibody. The method truly realizes a limit range of autonomous mutation of the antibody genes by performing gene editing on cells; and by combining a subsequent flow sorting technology, the method can directionally select antibody evolution and improve the efficiency of antibody evolution.

Description

technical field [0001] The present invention relates to the field of cell biology, in particular to an antibody evolution method and its application. Background technique [0002] Faced with complex living environments, immune cells in the human adaptive immune system can recognize different antigens or pathogens by diversifying their cellular receptors. For example, diverse B-cell receptors and T-cell receptors are the molecular basis for the recognition of various pathogens in B- or T-cell-mediated immune responses. B-cell receptors can be classified as membrane-bound or secreted, and secreted B-cell receptors are also known as antibodies. Antibody is an important molecule of humoral immunity. It consists of two identical heavy chains and light chains. The N-terminus is also called the variable region because of the diversity of amino acids in its composition. It can recognize and bind antigen molecules and determine the antibody specificity. The variable regions are li...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22C07K16/00C40B40/10
CPCC12N15/907C12N9/22C07K16/00C40B40/10C07K2317/92
Inventor 孟飞龙刘浏黄敏刘婷婷蔡燕妮
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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