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Reagent for cell infection by viruses and application of reagent

A virus infection and reagent technology, applied in the field of genetic engineering, can solve the problems of poor infection efficiency and complicated operation of T cells, and achieve the effect that the killing ability is not affected.

Active Publication Date: 2019-09-10
北京华奥玄德生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] For this reason, the object of the present invention is to overcome the shortcomings of poor efficiency and complicated operation in the prior art for infecting T cells, and provide an infectious reagent for infecting T cells and its application

Method used

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  • Reagent for cell infection by viruses and application of reagent
  • Reagent for cell infection by viruses and application of reagent
  • Reagent for cell infection by viruses and application of reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Expansion of primary T cells

[0029] Adopt the company's previous patent (application number 201810090357.1) amplification technology. It's as simple as:

[0030] 1. Activate the culture bottle: add the activator to the culture bottle until it covers the bottom of the bottle. 2 Add 10mL of activator to the cell culture flask to fill the bottom of the flask, and incubate at 4°C for 12 hours to obtain an activated culture flask. The activator is prepared by dissolving CD3 monoclonal antibody and IFN-γ in PBS buffer solution (pH 7.35-7.45). The concentration of CD3 monoclonal antibody was 50 μg / mL, and the concentration of IFN-γ was 2000 U / mL;

[0031] 2. Preparation of activation medium: Add 5mL of autologous plasma and 1mL of CIK cell activator to 44mL of basal medium to obtain an activation medium, wherein the CIK cell activator consists of IL-2, IL-1α and IFN-γ It is prepared by dissolving in PBS buffer solution, the concentration of IL-2 is 50000U / mL, th...

Embodiment 2

[0033] Example 2, the process of lentivirus production carrying CAR gene sequence

[0034] 1. 293T cell preparation:

[0035] Take out a frozen 293T cell (purchased from ATCC) from liquid nitrogen and quickly put it in a 37°C water bath until the ice cube disappears, add it dropwise to a 15ml centrifuge tube containing 5ml preheated medium, centrifuge at 1200rpm for 3min, discard Clear, resuspend the cells in 293T medium (10% FBS + 1mM sodium pyruvate + 2mM glutamine + 1% non-essential amino acids + DMEM) and inoculate them into 150mm culture dishes at 37°C, 5% CO 2 Cultivate in saturated humidity.

[0036] During the culture process, when the confluence of the cells reaches more than 90%, carry out subculture, discard the old medium, add 5ml sterilized PBS solution, shake gently, discard the PBS solution after washing the cells, and add 2ml 0.25% Trypsin-EDTA Digestion solution, digest for 1-2min until the cells are completely digested. Serum-containing medium was added to...

Embodiment 3

[0046] The infection efficiency of the EGFP virus of embodiment 3 different multiplicity of infection to primary generation T cell

[0047] The obtained high-titer lentiviral particles carrying the EGFP green fluorescent protein reporter gene were used to infect the obtained primary T cells, and the infection efficiency of different infection methods and different virus multiplicity on the primary T cells was explored.

[0048] The infection method is selected as follows: (1) directly add the virus into the T cell culture system for one infection; (2) directly add the virus into the T cell culture system for two infections, that is, replace the culture medium after the first infection, and then carry out one infection. (3) Centrifuge at 1000g at 4°C for 1h; (4) Centrifuge at 1000g at 32°C for 1h.

[0049] Multiplicity of Virus Infection We chose MOIs from 0-100 for infection, namely 0, 20, 40, 60, 80 and 100 MOIs. All groups were added 8 μg / ml polybrene as auxiliary infectio...

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Abstract

The invention discloses a reagent for cell infection by viruses and application of the reagent, and belongs to the technical field of gene engineering. The reagent for cell infection by the viruses comprises two or three of dinconazole nitrate, decitabine and sodium butyrate. By utilizing the reagent, the efficiency of infecting passage T cells by the lentiviruses can reach 80% or above, and is far higher than that of existing methods. Meanwhile, a carrier of CAR-T is adopted, the proliferation capability of CAR-T cells obtained by means of the technology is not influenced, and the killing capability on tumors with specificity is not influenced either.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a reagent for virus-infected cells and its application. Background technique [0002] Tumor immunotherapy is a treatment method that uses the immune system to treat cancer by restoring the anti-tumor immune response ability of the immune system, including monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy and small molecular inhibitors, etc. The current Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) and T-cell receptor (TCR) chimeric T cells (TCR-T) are two major technologies for adoptive cell reinfusion therapy. The latest immune cell technology is considered to be one of the most promising treatment methods in adoptive cell immunotherapy for tumors. [0003] The technical principle of T cell adoptive cell reinfusion therapy is as follows: the gene sequence of recombinant TCR and chimeric anti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
CPCC12N5/0636C12N15/86C12N2510/00C12N2740/15043
Inventor 段海峰薛冰华于婷婷解晶肖秀孝庞如梦弓景波张群伟
Owner 北京华奥玄德生物医药科技有限公司
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