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An expression vector, construction method and application of light-regulated T cell activation

An expression vector, light regulation technology, applied in biochemical equipment and methods, vectors, applications, etc., to achieve the effects of time and space controllability and fast light regulation

Active Publication Date: 2020-01-10
FUZHOU HOSPITAL FOR INFECTIOUS DISEASE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Therefore, in view of the significant role of T cells in tumor therapy, if it is possible to construct a light-induced T cell activation system, use light to induce T cells to kill tumors, and use T cells for immunotherapy of solid tumors, there may be unexpected effects

Method used

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  • An expression vector, construction method and application of light-regulated T cell activation
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  • An expression vector, construction method and application of light-regulated T cell activation

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Construction of OPN4 lentiviral expression vector

[0036] Entrusted Suzhou Jinweizhi Biotechnology Co., Ltd. to perform sequence synthesis (SEQ ID No.1 sequence), the synthesized sequence was added with 5'EcoRI and 3'BamHI restriction sites, and cloned into the pUC57 vector to obtain the recombinant vector pUC57-OPN4.

[0037] The synthetic recombinant vector was digested with EcoRI and BamHI to recover and purify the OPN4 gene sequence fragment, and the pCDH-CMV-MCS-EF1-Puro vector plasmid (purchased from Shanghai Jiran Biotechnology Co., Ltd.) was also digested with double enzymes to recover and purify the linear vector .

[0038] The linear vector and the OPN4 gene fragment were ligated at a ratio of 1:5 with T4 DNA ligase (Treasure Bioengineering (Dalian) Co., Ltd.) at 16°C, and the ligated product was transformed into competent E. 100 μg / ml) LB immobilized medium plate (upper, cultivate overnight at 37°C, pick a single colony in LB liquid medium (recip...

Embodiment 2

[0039] Example 2: Lentiviral packaging of OPN4 plasmid

[0040]HEK293T cells were inoculated in 10cm cell culture dishes with DMEM complete medium (DMEM medium containing 10% fetal bovine serum, 100U / ml penicillin, 100U / ml streptomycin), and when they grew to 70%-80% confluence, use Lipofectamin3000 transfection reagent transfects OPN4 lentiviral expression plasmid (pCDH-CMV-OPN4-EF1-Puro) and related lentiviral packaging plasmids (PLP-1, PLP-2 and pLP / VSVG), plasmid transfection amount: PLP-1 6.5 μg, PLP-2 2.5 μg, pLP / VSVG 3.5 μg, pCDH-CMV-OPN4-EF1-Puro 10 μg. After culturing for 12 hours, replace the fresh cell culture medium; continue culturing for 24 hours, collect the virus supernatant by centrifugation, filter through a 0.45 μm filter membrane, and store the obtained virus liquid at -80°C for subsequent infection of cells. (Lentiviral packaging plasmids PLP-1, PLP-2 and pLP / VSVG were purchased from Shanghai Jiran Biotechnology Co., Ltd.).

Embodiment 3

[0041] Example 3: Recombinant lentivirus infection of 293T cells or Jurkat T cells

[0042] 293T cells were inoculated into 6-well plates until they reached 60-80% confluence, and 1.5ml of fresh DMEM complete medium and 0.5ml of virus solution were added;

[0043] Jurkat T cells 8 x 10 5 Each was resuspended with 1ml 1640 complete medium (1640 medium containing 10% fetal bovine serum, 100U / ml penicillin, 100U / ml streptomycin), and 1ml virus liquid was added;

[0044] Both cell lines were added Polybrene to a final concentration of 4 μg / ml, mixed gently, and placed in an incubator for 12 hours. The culture medium containing the virus was absorbed, and fresh DMEM complete medium (293T cells) or 1640 complete medium ( Jurkat T cells). After continuing to culture for 48 hours, change to fresh DMEM complete medium or 1640 complete medium containing 3 μg / ml Puromycin to screen for stably transduced cell lines, and obtain 293T cells and Jurkat T cells stably expressing OPN4 after s...

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Abstract

The invention relates to an expression vector capable of optically regulating and controlling T cell activation, a construction method, application and a method for activating T cells through the expression vector. The expression vector capable of optically regulating and controlling T cell activation is obtained by inserting blue-light-inductive protein OPN4 shown in SEQ ID No.1 into a slow virus expression vector pCDH-CMV-MCS-EF1-Puro. The expression vector, the construction method, the application and the method have the main advantages that the expression vector which can induce blue light and activate the transcriptional activity of a NFAT can effectively control transcriptional activation of the NFAT through the blue light to induce activation of T cells. A novel convenient and controllable though is provided for enhancement on the effect of immunotherapy by activating the T cells in clinic, and compared with traditional T cell activation induced by chemical drugs, optical regulation and control are faster and more reversible, and the time and space controllability is achieved.

Description

[0001] (1) Technical field [0002] The invention relates to an expression vector for light-regulated T cell activation, a construction method and application thereof, and a method for activating T cells by using the expression vector. [0003] (2) Background technology [0004] Both synthetic biology and optogenetics are frontier biological fields that have just emerged in recent years. Light is one of the most common and easily available substances in nature. Compared with traditional chemical small molecule inducers, light inducers are not only cheap and easy to obtain, but also can be precisely regulated in time and space. Therefore, using light Biologists have been pursuing the goal of regulating gene expression as an inducer, and then regulating various metabolic activities of living organisms. However, previous methods are seldom used due to technical complexity and limitations. In recent years, with the advancement of molecular biology experiment technology, a system ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/10C12N15/66
CPCC12N15/66C12N15/86C12N2740/15043C12N2800/107
Inventor 刘小龙赵必星刘景丰谭雄红
Owner FUZHOU HOSPITAL FOR INFECTIOUS DISEASE
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