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Preparation of T-lymphocyte preparations in which the PD-1 immune checkpoint is de-suppressed

An immune checkpoint and PD-1 technology, applied in the field of molecular biology, can solve the problems of large side effects, complicated preparation process, and long cycle of T lymphocyte preparations, and achieve the effects of avoiding side effects, simple preparation process, and short production cycle

Inactive Publication Date: 2021-03-26
王清路 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The T lymphocyte preparations used for tumor treatment in the prior art have large side effects, complicated preparation process and long cycle

Method used

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  • Preparation of T-lymphocyte preparations in which the PD-1 immune checkpoint is de-suppressed
  • Preparation of T-lymphocyte preparations in which the PD-1 immune checkpoint is de-suppressed
  • Preparation of T-lymphocyte preparations in which the PD-1 immune checkpoint is de-suppressed

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Experimental program
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Embodiment 1

[0059] (1) Construction of single-base gene editing PD-1 transfection system

[0060] ① Design of gRNA target sequence

[0061] Obtain the full gene sequence of PD-1 (EF064716.1) from NCBI, find the gene sequence corresponding to the ITSM region, design the gRNA target sequence based on the CRISPR-DO website, and then select a suitable target sequence 5'-GTGGCATACTCCGTCTGCTC according to the set parameters -3' (20bp length), then the gRNAtarget sequence was synthesized by Hanbio Technology (Shanghai) Co., Ltd., and the sticky end was added to form a double-stranded oligo sequence.

[0062] The pHBLV-U6-gRNA-EF1-ZsGreen plasmid ( figure 2 ) for enzyme digestion, and the digested product was subjected to agarose gel electrophoresis to detect the effect of the enzyme digestion, and the target vector band was cut from the gel after agarose gel electrophoresis, and the gel was recovered to obtain a linearized expression vector.

[0063] Use T4 ligase to ligate the gRNA target se...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a preparation method of a T lymph cell preparation for removing restraint of PD-1 immunity checkpoints. The preparation method comprises the steps of constructing a single-basic-group gene editing PD-1 transfection system, performing T lymph cell separation and activation, performing T lymph cell cotransfection, performing screening with a flow cytometer and sequencing analysis, and performing identification. According to the preparation method disclosed by the invention, key tyrosine site codon mutation of ITSM region gene sequence of human PD-1 is realized, and removal of immunity checking function of PD-1 protein after genetic transcription and translation is caused. The T lymph cell preparation for removing restraint activity of the PD-1 immunity checkpoints, prepared by the preparation method disclosed by the invention can promote the capacity of the T lymph cells for destroying tumor cells, reserve the biological activity and other functions of PD-1 greatly and strengthen long-term tumor immunity effects.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for preparing a T lymphocyte preparation that releases inhibition of a PD-1 immune checkpoint. Background technique [0002] PD-1 (Programmed Death 1, programmed death receptor-1) is an important immunosuppressive molecule. The pathway composed of PD-1 and its ligands PD-L1 and PD-L2 plays a crucial role in maintaining peripheral immune tolerance. Tumors and pathogens that cause chronic infections can use this pathway to escape from T cell-mediated tumor-specific and pathogen-specific immunity, mainly through the production of PD-L1 on its surface, when PD-L1 is linked to a type of immune cell T The PD-1 protein on the cells inactivates the T cells, and the T cells cannot find the tumor and send a signal to the immune system to attack the tumor. The PD-1–PD-L1 co-inhibitory pathway has been used in the development of tumor therapy and has achieved...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/113C12N15/90
CPCC07K14/70521C12N5/0636C12N15/113C12N15/907C12N2310/10C12N2310/20
Inventor 王清路
Owner 王清路