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Method for rapidly identifying high-affinity TCR antigenic cross-reaction activity

A cross-reactive, high-affinity technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., to avoid false positives, show increased kurtosis, and simplify the operation process.

Active Publication Date: 2021-03-16
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these technologies are all TCR or pMHC for yeast display, and the pMHC or TCR in the other direction exists in the form of tetrameric proteins for interaction screening, and there are no reports on combining with yeast mating technology to study protein interactions

Method used

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  • Method for rapidly identifying high-affinity TCR antigenic cross-reaction activity
  • Method for rapidly identifying high-affinity TCR antigenic cross-reaction activity
  • Method for rapidly identifying high-affinity TCR antigenic cross-reaction activity

Examples

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Embodiment 1

[0067] This example mainly introduces a method for rapidly identifying the cross-reactivity of high-affinity TCR antigens.

[0068] as attached figure 1 As shown, a method for rapidly identifying high-affinity TCR antigen cross-reactivity, comprising the following steps:

[0069] S1. Adjust the TCR and pMHC sequences to constitutive promoters to induce expression, integrate and express the elements into the yeast genome, and display the TCR and pMHC proteins on the surface of yeast with different mating types;

[0070] S2. Yeast mating experiments using different mating-type yeasts integrating TCR and pMHC;

[0071] S3, diploid screening and next-generation sequencing.

[0072] Further, the step of S1 includes:

[0073] Firstly, construct the TCR sequence to be studied into scTCR form, and obtain the peptide mutation library of pMHC;

[0074] Transform the pMHC library to construct the required receiving vector ysynalpha_DEST, and obtain the vector of ysyna-TCR and ysynalp...

Embodiment 2

[0107] This embodiment is carried out on the basis of the above-mentioned embodiment 1, and the similarities with the above-mentioned embodiment 1 will not be repeated.

[0108] This example mainly introduces a specific implementation method of a method for rapidly identifying high-affinity TCR antigen cross-reactivity, including the following steps:

[0109] S1. Adjust the TCR and pMHC sequences to constitutive promoters to induce expression, integrate and express the elements into the yeast genome, and display the TCR and pMHC proteins on the surface of yeast with different mating types;

[0110] S2. Yeast mating experiments using different mating-type yeasts integrating TCR and pMHC;

[0111] S3, diploid screening and next-generation sequencing.

[0112] Further, the step of S1 includes:

[0113] Firstly, construct the TCR sequence to be studied into scTCR form, and obtain the peptide mutation library of pMHC;

[0114] Transform the pMHC library to construct the required...

Embodiment 3

[0149] This example mainly introduces the element design for the integrated expression of TCR and pMHC.

[0150] This embodiment is carried out on the basis of the above-mentioned embodiment 1, and the similarities with the above-mentioned embodiment 1 will not be repeated.

[0151] In the present study, the yeast surface display of TCR and pMHC utilized the α-lectin-target gene surface display system. α-lectin is composed of core subunit Aga1 and binding subunit Aga2, which are connected by disulfide bonds, and the C-terminus of core subunit Aga1 is covalently bound to cell wall glucan. TCR and pMHC are fused to the C-terminal or N-terminal of the Aga2 protein in yeast a-lectin, the expression of the fusion protein is induced by the GAL1 promoter, which is a galactose-inducible system, and the yeast grows in glucose medium When the transcription of the GAL1 promoter is completely inhibited, the Aga2p fusion gene product can be induced to produce the Aga2p fusion gene product...

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Abstract

The invention relates to a method for rapidly identifying high-affinity TCR antigenic cross-reaction activity. According to the invention, TCR and pMHC sequences are adjusted into constitutive promoter for inducible expression, and the element is integrated into into a yeast genome to be expressed. The method comprises the following steps: displaying TCR and pMHC proteins on the surfaces of yeastsof different mating types; carrying out a yeast mating experiment by utilizing TCR and pMHC-integrated yeasts of different mating type ; and carrying out diploid screening and next generation sequencing. According to the method for rapidly identifying high-affinity TCR antigenic cross-reaction activity provided by the invention, pure protein is not needed, only a DNA sequence needs to be synthesized, and a more complete antigen mutation library can be obtained within one week by using a Golden-gate cloning method, thus being convenient and efficient; and the limitation of screening on the cellular level is broken through by means of a yeast mating system, operation steps are simplified, an antigen sequence interacting with a specific TCR can be obtained in a short time, and experimental results are reliable.

Description

[0001] technology domain [0002] The invention relates to the field of biotechnology, in particular to a method for quickly identifying the cross-reactivity of high-affinity TCR antigens. Background technique [0003] TCR is a molecule on the surface of T cells that specifically recognizes antigens and mediates immune responses. In the adaptive immune system, TCR specifically recognizes the antigen molecule polypeptide (pMHC) presented by histocompatibility complex molecules, thereby activating T cells and producing immune effects. Tumor cell immunotherapy-TCR-T therapy is a treatment method that uses T cells that specifically bind to the target tumor antigen to kill malignant cells. The high-affinity TCR evolved in vitro can improve the anti-tumor ability of transformed T cells, but it also recognizes non-target antigens due to the problem of antigen cross-reactivity, causing serious side effects to patients. [0004] Currently, one of the methods for studying the cross-re...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12N1/19C12N15/12
CPCC12Q1/6869C07K14/7051C07K14/70539C12Q2531/113C12Q2535/122C12Q2525/191
Inventor 蓝勋王莉慧
Owner TSINGHUA UNIV
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