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NK (Natural Killer) trophoblast cell for expressing cytokine composition as well as preparation method and application of NK trophoblast cell

A technology of trophoblast cells and cells, applied in the field of NK trophoblast cells and their preparation

Pending Publication Date: 2021-05-11
HENAN HUALONG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the efficiency of amplification and activation needs to be further improved

Method used

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  • NK (Natural Killer) trophoblast cell for expressing cytokine composition as well as preparation method and application of NK trophoblast cell
  • NK (Natural Killer) trophoblast cell for expressing cytokine composition as well as preparation method and application of NK trophoblast cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 lentiviral vector construction

[0060] Whole gene synthesis tandem CD19 coding sequence and CD86 coding sequence KCD19CD86 (SEQ ID NO:6), IL-21-CD8α coding sequence KIL21CD8α (SEQ ID NO:7), CD64 coding sequence KCD64 (SEQ ID NO:8), CD137L Coding sequence (SEQ ID NO:9), and restriction site BamHI / EcoRI or BamHI / NotI are added at both ends;

[0061] Prepare the enzyme digestion reaction system shown in Table 1, digest the synthetic gene in a water bath at 30°C for 1 hour and a water bath at 37°C for 1.5 hours, and then connect it into the linearized lentiviral vector pCDH digested with the same restriction endonuclease ;

[0062] Table 1

[0063] components volume Ultra-pure water 16μL BamHI 2.5μL EcoRI / NotI 2.5μL 10×K Buffer 2.5μL 0.1%BSA 5μL dna 22μL (5μg)

[0064] The digested products were detected by agarose gel electrophoresis. The electrophoresis condition was 120V constant current electroph...

Embodiment 2

[0065] Example 2 Recombinant lentivirus packaging and titer detection

[0066] In this example, the calcium phosphate transfection method (Beyotime-Calcium Phosphate Cell Transfection Kit, product number C0508) was used to co-transfect 293T cells with the lentiviral expression vector and the packaging plasmid, and the lentivirus was packaged, and the centrifugal ultrafiltration method (Millipore-Centrifugal Filter Units Amicn (R) Ultra-15, product number UFC910096, 100kD) for collection and concentration of lentivirus, the steps are as follows:

[0067] (1) 24 hours before transfection, digest 293T cells in the logarithmic growth phase with 0.125% trypsin (the degree of cell confluence is 85-95%), and use complete medium DMEM containing 10% fetal bovine serum + 1mg / mL double antibody Adjust the cell density and passage, re-seeded in the d15 culture dish, 37 ℃, 5% CO 2 , cultivated under saturated humidity;

[0068] (2) After 24 hours, replace the cell culture medium with fre...

Embodiment 3

[0077] Construction and identification of embodiment 3 NK trophoblast cells

[0078] Inoculate 3mL of K562 cell suspension in a good growth state into a 60mm culture dish, add the packaged recombinant lentivirus to K562 cells according to the ratio of MOI=100:1, and add polybrene (8μg / mL) to carry out lentiviral Transfection, puromycin selection to obtain K562 expressing CD19, CD86, IL21, CD64 and CD137L, as NK trophoblast cells;

[0079] take 10 7 Add 1mL Trizol to NK trophoblast cells, blow and mix with a pipette tip to promote cell lysis, and place at room temperature for 5min; add 0.2mL chloroform, mix by inverting repeatedly for 15s, centrifuge at 12000g for 10min at 4°C; take the upper aqueous phase in a new Add 0.5mL isopropanol to the centrifuge tube, mix gently, place at room temperature for 10min, centrifuge at 12000g at 4°C for 10min; discard the supernatant, add 1mL of 75% alcohol to gently wash the precipitate, and centrifuge at 12000g at 4°C for 10min; Discard ...

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Abstract

The invention provides an NK (Natural Killer) trophoblast cell for expressing a cytokine composition as well as a preparation method and application of the NK trophoblast cell. The NK trophoblast cell expresses membrane proteins CD19, CD86, IL21, CD64 and CD137L; the CD19 comprises an amino acid sequence as shown in SEQ ID NO:1; and the CD86 comprises an amino acid sequence as shown in SEQ ID NO:2. The NK trophoblast cell constructed by the invention has high expression rate on CD19, CD86, IL21, CD64 and CD137L, and the multiplication capacity of the NK cells is synergistically activated by the factors, so that the effect of inducing the NK cells to differentiate and mature in vitro is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a NK trophoblast cell expressing a cytokine composition and a preparation method and application thereof. Background technique [0002] Natural killer cells (NK) are important immune cells, not only the body's first line of defense against cancer cells and virus infection, but also have immune regulation function. Clinical studies have found that NK cell adoptive immunotherapy for malignant tumors has a good application prospect. [0003] The existing NK cell culture methods mainly include factor induction method, antibody coating method, special NK medium culture method and stem cell induction method. Among them, the factor induction method is to culture NK cells in peripheral blood mononuclear cells (PBMC) in ordinary lymphocyte culture medium, and add cytokines and / or feeder cells; the antibody coating method is to culture NK cells coated with antibodies. Ordinary lymphocyte mediu...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/62C12N15/12C12N5/0783
CPCC12N5/0694C12N15/86C07K14/70503C07K14/70532C07K14/70517C07K14/54C07K14/70535C07K14/70578C12N5/0646C12N2510/00C12N2740/15043C12N2800/107C07K2319/03C12N2502/99
Inventor 赵礼军熊建民王蓓蕾
Owner HENAN HUALONG BIOLOGICAL TECH
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