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A gene and transfection vector for transfecting NK cells

A kind of NK cell and gene technology, applied in the field of genetic engineering, can solve problems such as acute or chronic allergic reactions and strong killing ability

Active Publication Date: 2020-10-09
BEIJING DCTY BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, since NK cells transfected with IL-15 can proliferate indefinitely, they are potentially tumorigenic
NK cells have strong killing ability, and NK cell therapy may also cause acute or chronic allergic reactions

Method used

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  • A gene and transfection vector for transfecting NK cells
  • A gene and transfection vector for transfecting NK cells
  • A gene and transfection vector for transfecting NK cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The gene for transfection of NK cells: ICasp9-T2A-IL15-T2A-NGFR-T2A-the target gene of interest (the target gene of interest such as GFP green fluorescent protein gene) is subcloned into the mammalian cell expression vector pCMV. Through gene synthesis, the NheI restriction site flanking sequence GCTAGC was added to the 5' end of the gene during synthesis, and the XbaI restriction site flanking sequence TCTAGA was added to the 3' end of the gene. NheI and XbaI 10U (Neb Company) were used to digest the synthesized gene fragment and 1 μg of pCMV plasmid in 20 μl Cutsmart system for 1 h each.

[0045] The digested product was subjected to 1% agarose gel electrophoresis, and a single band was cut out for gel recovery. Take 50ng of the recovered gene fragment and pCMV plasmid respectively, add 4000 units of T4 ligase (ABclonal Company), make up the volume to 20 μl with pure water, and ligate at room temperature for 2 hours to obtain a vector for transfection of NK cells. Ta...

Embodiment 2

[0052] Compared with NK92, the morphological changes of the 915NGFR cells obtained in the embodiment when the interleukin was removed from culture:

[0053] experimental method:

[0054] Cell culture: 5 × 10 each for NK92 and 915NGFR 5 Cells were cultured in 1ml LY-08 medium, supplemented with 10% FBS, 1% double antibody, 1% BioMyc-3, no IL-2, cell incubator at 37°C, 5% CO 2 , replace the medium every 24h, adjust the cell density to 5×10 5 pieces / ml. Take 20 μl of cells for AOPI staining, measure the cell diameter with a cell counter, and record the continuous detection for 4 days. The results are shown in Table 1 and figure 2 .

[0055] Table 1 Comparison table of cultured cell diameter

[0056]

[0057] From Table 1 and figure 2 It can be concluded that in culture, 915NGFR can still proliferate rapidly in the absence of IL-2, and the cell diameter remains unchanged. The NK92 cells gradually shrank and died.

Embodiment 3

[0059] Compared with NK92, the 915NGFR cells obtained in Example 1 remove the changes in quantity and viability of interleukin culture:

[0060] experiment method:

[0061] 5×10 for NK92 and 915NGFR respectively 5 Cells were cultured in 1ml LY-08 medium, supplemented with 10% FBS, 1% double antibody, 1% BioMyc-3, no IL-2, cell incubator at 37°C, 5% CO 2 , replace the medium every 24h, adjust the cell density to 5×10 5 pieces / ml. Take 20 μl of cells for AOPI (Nexcelom, CS2-0106-5ml) staining, count the number of cells with a cell counter (Nexcelom, CellometerK2), and record the cell viability. Continuous detection records for 4 days, see the results image 3 with Figure 4 .

[0062] from image 3 with Figure 4 It can be seen from the figure that in the change of the number of cell proliferation, 915NGFR can still proliferate rapidly in the absence of IL-2, the number increases, and the survival rate remains basically unchanged, while NK92 cells cannot continue to prol...

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Abstract

The invention provides a gene transfected with NK cells and a transfection vector, which belongs to the technical field of gene engineering, and is characterized in that an ICasp9 gene, a T2A gene, anIL15 gene, a T2A gene, an NGFR gene, a T2A gene and a target gene are sequentially connected in series. The gene transfected with the NK cell provided by the invention is connected to a pCMV vector,the vector transfected with the NK cells is obtained; after the vector is transfected with NK cells, drug screening and flow screening are carried out, the NK cell expressing the target gene is obtained, the NK cell expressing the target gene can simultaneously express a switch structure, a suicide reaction can be caused when an inducer is added, the long-term existence of the NK cell expressing IL-15 is prevented, and the intervention can be performed when the 915NGFR cell has the side effect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gene for transfecting NK cells and a transfection carrier. Background technique [0002] NK cells account for about 15% of circulating blood lymphocytes in normal adults and are a kind of innate immune cells. NK cells recognize and clear pathogen-infected and mutated cells. NK cells, as a kind of innate immune effector cells, have shown a strong ability to kill target cells since they were first reported in 1975. Due to the limited proliferative ability of primary NK cells, the use of NK cell lines gets rid of the expensive NK in vitro culture system and reduces the production time, so it is widely used in clinical practice. Among them, NK-92 cells were isolated from a white male in 1992. They are the easiest NK cell line for gene editing and modification, and are widely used in clinic. The proliferation of NK92 depends on higher concentrations of int...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/85
CPCC07K14/43595C07K14/5443C07K14/71C12N9/22C12N15/85
Inventor 焦顺昌张嵘袁翰
Owner BEIJING DCTY BIOTECH CO LTD
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