Gene, vector and method for preparing immortalized dendritic cells and immortalized dendritic cells

A dendritic cell and immortalization technology, applied in the field of genetic engineering, can solve the problems of low presentation ability and limited application range

Active Publication Date: 2020-05-01
BEIJING DCTY BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the existing immortalized DC technology can solve the above-mentioned problem of low presentation ability, it is only suitable for patients with HLA-0201, and its application range is limited

Method used

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  • Gene, vector and method for preparing immortalized dendritic cells and immortalized dendritic cells
  • Gene, vector and method for preparing immortalized dendritic cells and immortalized dendritic cells
  • Gene, vector and method for preparing immortalized dendritic cells and immortalized dendritic cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] plasmid extraction

[0053] 1) Shaking protocol: Melt Glycerol bacteria on ice, Glycerol bacteria contain the vector P-ST40-TAX2 for preparing immortalized dendritic cells (the vector contains ST40 gene and TAX2 gene), take 100 μL to 3 mL each containing 100 μg / mL In the LB medium of ampicillin, activate at 37°C and 220rpm for 6h. The activated bacterial liquids were respectively expanded into 100 mL LB medium containing 100 μg / mL ampicillin, and cultured overnight at 37° C. and 220 rpm. The next day, centrifuge at 4000×g for 10 minutes to collect the sediment of each bacterial solution.

[0054] Among them, glycerol bacteria: after transforming the competent cell Stbl3 with the vector containing ST40 gene and TAX2 gene, pick the monoclonal shaking bacteria, discard the supernatant after the bacterial solution is centrifuged, keep the bacterial sediment, add 25% glycerol to weigh the bacterial sediment Suspended to obtain glycerol bacteria. Competent cells Stbl3 were...

Embodiment 2

[0082] Isolation and Activation of Dendritic Cells from Peripheral Blood by Adherence Method (DC)

[0083] 1) Blood collection and separation of PBMC;

[0084] 2) Adjust the PBMC to 1×10 medium 1640+10% FBS 6 Cells / mL, as in Petri dish, at 37°C 5% CO 2 Incubator, rest overnight; collect suspension cells, mark as T+B cells, and set aside;

[0085] 3) Pipette the cells attached to the bottom of the culture dish with medium 1640+10% FBS+100IU / mL IL-2, and collect the adherent mononuclear cells, which are peripheral blood-derived DCs,

[0086] 4) Adding 35 μg / mL of PHA, stimulating and culturing the cells for 24 hours, and then preparing for use to obtain dendritic cells.

Embodiment 3

[0088] Construct lentiviral vector and transform DC

[0089] 1) Packaging lentivirus: 293T cell plating density: 1.8×10 7 , 20mL OPTI-MEM medium placed at 37 °C 5% CO 2 to cultivate. The amount of transfection plasmid obtained in Example 1: the amount of packaging plasmid: 15 μg, P-ST40-TAX2 plasmid (plasmid for preparing immortalized dendritic cells): 15 μg, adding the plasmid into the buffer and shaking and mixing for 5 seconds, adding the transfection Transfection reagent: 60 μL, pipette to mix 5 times, incubate at room temperature for 10 minutes, evenly distribute the transfection mixture into the cells, 37°C 5% CO 2 to cultivate. 3 hours after transfection, fresh medium was replaced, and 37 mL of OPTI-MEM (6% FBS) medium was added to each T175 culture bottle, and the supernatant was harvested at 96 hours for later use.

[0090] 2) DC infected with lentivirus: use 1mL 1640+10%FBS+0.1%polybrene+100μL virus supernatant to resuspend the activated DC obtained in Example 2,...

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Abstract

The invention provides a gene, a vector and a method for preparing immortalized dendritic cells and the immortalized dendritic cells, belonging to the technical field of genetic engineering. The genefor preparing the immortalized dendritic cells is obtained by connecting an ST40 gene with a TAX2 gene through a linker, wherein the nucleotide sequence of the ST40 gene is as shown in SEQ ID No. 1; and the nucleotide sequence of the TAX2 gene is shown as SEQ ID No. 2. The immortalized dendritic cells prepared from the genes provided by the invention comprise HLA-A0201, and covers nearly nine types of common HLA-A1101, HLA-A2402, HLA-A0301 and the like of Chinese population, so the application range is greatly enlarged.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gene, a carrier, a method for preparing immortalized dendritic cells and the immortalized dendritic cells. Background technique [0002] Adoptive cell therapy with T lymphocytes as the main body plays an increasingly significant role in anti-tumor immunotherapy, and how to obtain a sufficient number of antigen-specific T cells has become the most important technical difficulty at present. The traditional expansion method of specific T lymphocytes is mainly the co-cultivation of antigenic peptides and PBMCs of patients, which mainly relies on the presentation of antigen-presenting cells naturally present in PBMCs, especially the presentation of DCs, and then activates T cells; natural DCs in The survival time of in vitro culture is short, the proportion in the patient's PBMC is low, and the presentation ability is limited. Although the existing immortali...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K14/47C12N15/867
CPCC07K14/47C07K2319/00C12N15/86C12N2740/15043
Inventor 焦顺昌张嵘陈寅
Owner BEIJING DCTY BIOTECH CO LTD
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