A recombinant plasmid co-expressing NFAT and human dnam-1 protein, recombinant cell and its construction method and application

A DNAM-1, recombinant plasmid technology, applied in the field of biomedicine, can solve the problem of not being able to obtain the molecular antibody that activates DNAM-1, and achieves the effect of simple operation

Active Publication Date: 2022-03-22
BEIJING DCTY BIOTECH CO LTD
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Problems solved by technology

[0003] In the past, the method of detecting DNAM-1 activation was mainly to use the method of regulating the antibody CD155 to develop therapeutic antibodies. However, because CD155 is a competitive receptor, this method can not only bind to CD226 protein to activate NK cells, but also It can bind to TIGIT protein to inhibit the function of NK cells, and cannot activate DNAM-1 molecular antibody

Method used

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  • A recombinant plasmid co-expressing NFAT and human dnam-1 protein, recombinant cell and its construction method and application
  • A recombinant plasmid co-expressing NFAT and human dnam-1 protein, recombinant cell and its construction method and application
  • A recombinant plasmid co-expressing NFAT and human dnam-1 protein, recombinant cell and its construction method and application

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preparation example Construction

[0036] The present invention also provides a method for preparing the above-mentioned recombinant cell Jurkat-NFAT-Luc-CD226, comprising the following steps: using the above-mentioned recombinant plasmid PB-NFAT-Luc-CD226 to transfect Jurkat cells to obtain the recombinant cell J u rk a t-NFAT-L uc -CD226.

[0037] The transfection of the present invention is preferably carried out according to the instructions of the electrotransfer instrument Celetrix, CTX-1500A), specifically comprising: using Jurkat cells with fetal bovine serum-free (REF: 10099-141, brand: gibco), penicillin-streptomycin-free mixed solution (Cat: SV30010 Brand: HyClone) was washed and centrifuged with 1640 medium, then resuspended with 120 μl of 1640 medium (no fetal bovine serum, no penicillin-streptomycin mixture), added the plasmid and mixed gently, then transferred the cell suspension Put it into the electric shock cup (120μl Celetric Cat.NO.1204) that is matched with the electrotransfer instrument,...

Embodiment 1

[0051] Example 1J u rk a t-NFAT-L uc - Construction of CD226 cells

[0052] 1. Protocells

[0053] The original Jurkat cells (Clone E6-1) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, subcultured and preserved in the laboratory, and the cell viability of the cells used was over 95%.

[0054] 2. Plasmid construction

[0055]NFAT-reverse was synthesized by whole gene synthesis technology, and a SpeI restriction site was added to its 5' end, and a BstBI restriction site was added to the 3' end, and then the synthesized NFAT-reverse gene (SEQ ID NO.1) was passed The upstream SpeI restriction site and the downstream BstBI restriction site were constructed into the vector PB-713B-1 (purchased from SBI Company, LOT: 190316-004), and a small preparation of endotoxin-free plasmid was carried out (entrusted by Jinweizhi Biotechnology Co., Ltd. Ltd.) to obtain the PB-NFAT-Luc-713 plasmid.

[0056] Using humans as the host, the DNAM-1 gene sequence w...

Embodiment 2

[0066] Example 2 Jurkat-NFAT-Luc-CD226 monoclonal preparation and identification

[0067] 1. Using the successful Jurkat-NFAT-Luc-CD226 cell clone constructed in Example 1, the clone number is 35B8. Operate with reference to the flow cytometer (SONY) manual, take Jurkat-NFAT-Luc-CD226 and Jurkat-WT cells (negative control) and wash away the medium with PBS containing 0.5% BSA, then add PE anti-human CD226 ( DNAM-1) antibody, incubated at room temperature in the dark for 20min. After washing away the fluorescent antibody with PBS, resuspend to 1×10 6 / ml cell concentration, flow cytometry monoclonal DNAM-1 positive cells. The following method was used for verification, and the cells after monocloning the cells with high expression of NFAT reporter element and DNAM-1 protein were named Jurkat-NFAT-Luc-DNAM-1 monoclonal cells.

[0068] 2. Identification of DNAM-1 protein expression in Jurkat-NFAT-Luc-DNAM-1 monoclonal cells

[0069] Take 1×10 Jurkat-NFAT-Luc-DNAM-1 monoclonal...

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Abstract

The invention provides a recombinant plasmid co-expressing NFAT and human DNAM‑1 protein, a recombinant cell and its construction method and application, and relates to the technical field of biomedicine. The invention provides a method for constructing the recombinant plasmid and the constructed recombinant plasmid, and constructs recombinant cells and monoclonal recombinant cells based on the recombinant plasmid. The bidirectional promoter of the recombinant plasmid described in the present invention promotes the expression of the DNAM-1 gene and the NFAT-Luciferase gene, and the gene expression does not affect each other. The recombinant cells of the present invention are constructed based on Jurkat, a human immune type cell, which is closer to the NK cell model, and when constructing the recombinant cells, no virus is introduced, nor does it cause inflammation-related pathways in cells, and the operation is simple. Anti-human DNAM‑1 antibodies can also be detected and / or screened using the recombinant cells or monoclonal recombinant cells of the present invention.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a recombinant plasmid co-expressing NFAT and human DNAM-1 protein, a recombinant cell, a construction method and application thereof. Background technique [0002] DNAM-1 (DNAX accessory molecule-1, CD226, one of the activating receptors of NK cells), originally known as T cell lineage-specific activating antigen (TLISA1), is involved in regulating the differentiation of cytotoxic T cells (CTL). Later, it was found that it was also expressed on platelets and could participate in the activation and aggregation of platelets, so it was renamed as platelet T cell activation antigen (PTA1). Further research found that it was also expressed on the surface of NK cells. Formally named DNAM-1, a protein with multiple Ig-like domains that binds to MHC-I (major histocompatibility complex) molecules. It has been confirmed that DNAM-1 can positively regulate the activation and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/12C12N15/66C12N5/10C07K16/18G01N33/68
CPCC07K14/47C12N15/85C07K16/18G01N33/68C12N2800/22
Inventor 焦顺昌张嵘袁翰钟宏东
Owner BEIJING DCTY BIOTECH CO LTD
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