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Preparation method of PD-1 (Programmed cell death protein 1) and CTLA4 (Cytotoxic T-Lymphocyte Antigen 4) double-gene defect type T lymphocyte preparation

A technology of lymphocytes and double genes, applied in the field of molecular biology, can solve problems such as the difficulty of triggering T cell immune responses, achieve the effects of enhancing long-term tumor immunity, simplifying production procedures, and improving the ability of

Inactive Publication Date: 2017-11-24
山东百福基因科技有限公司 +1
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Problems solved by technology

In most cases, T cell surface antigen receptor (TCR) signal transduction can activate antigen-activated T cells, however, it is difficult to trigger T cell immune response solely by TCR signal transduction pathway

Method used

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  • Preparation method of PD-1 (Programmed cell death protein 1) and CTLA4 (Cytotoxic T-Lymphocyte Antigen 4) double-gene defect type T lymphocyte preparation
  • Preparation method of PD-1 (Programmed cell death protein 1) and CTLA4 (Cytotoxic T-Lymphocyte Antigen 4) double-gene defect type T lymphocyte preparation
  • Preparation method of PD-1 (Programmed cell death protein 1) and CTLA4 (Cytotoxic T-Lymphocyte Antigen 4) double-gene defect type T lymphocyte preparation

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Embodiment 1

[0042] (1) Construction of PD-1 and CTLA4 double gene knockout vector

[0043] ① Design of gRNA target sequence

[0044]Obtain the full gene sequence of PD-1 (EF064716.1) and CTLA4 (NG_011502.1) from NCBI, design the gRNA target sequence based on the CRISPR-DO website, and then select the appropriate target sequence for the two genes according to the set parameters (each 20bp in length), and then two complementary target sequences were synthesized by Shanghai Sangon Bioengineering Co., Ltd. (the sticky ends after BbsI digestion were added).

[0045] For example: PD-1 oligo: F 5’-ATAG CCGAGCCTACCATATGGTGT-3’

[0046] R 5'-AAAT ACACCATATGGTAGGCTCGG-3'

[0047] CTLA4 oligo: F 5'-ATAG CCGCTGCCAGAGATCCCAAA-3'

[0048] R 5'-AAAT TTTGGGATCTCTGGCAGCGG-3'

[0049] ② Knockout vector construction

[0050] The pU6gRNA-CMV-Cas9-GFP expression vector was digested with BbsI, and after recovery, it was ligated with the oligo double strand formed by the PD-1 oligo sequence to construct a ...

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Abstract

The invention belongs to the technical field of molecular biology and in particular relates to a preparation method of a PD-1 (Programmed cell death protein 1) and CTLA4 (Cytotoxic T-Lymphocyte Antigen 4) double-gene defect type T lymphocyte preparation. Construction of a PD-1 and CTLA4 double-gene knockout vector, T lymphocyte separation and activation, electric transfection of T lymphocytes and T7E1 enzyme digestion identification, and flow cytometer screening and sequencing analysis are carried out. According to the production of a gene defect type immune cell preparation, on one hand, a production procedure of cells is simplified; on the other hand, the capability of killing tumor cells by the T lymphocytes is improved and the long-period tumor immunity can be enhanced.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a preparation method of a PD-1 and CTLA4 double gene-deficient T lymphocyte preparation. Background technique [0002] T cells primarily recognize antigenic peptides presented by the major histocompatibility complex (MHC) on the cell surface. In most cases, T cell surface antigen receptor (TCR) signal transduction can activate antigen-activated T cells, however, it is difficult to trigger T cell immune response only relying on TCR signal transduction pathway. Therefore, efficient activation and functional mediation of T cells requires the synergy of dual signals: one is the antigenic peptide-MHC complex, which is recognized by the T cell antigen receptor (TCR); APC) and co-stimulatory molecule ligand and receptor pairs on the surface of T cells are provided. Among them, PD-1 / PD-L1 is recognized as the most basic co-stimulatory signal, which is produced by ...

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
CPCC12N15/85C12N5/0636C12N2510/00
Inventor 王清路
Owner 山东百福基因科技有限公司
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