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NK trophoblast cell as well as preparation method and application thereof

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

Pending Publication Date: 2021-05-28
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 trophoblast cell as well as preparation method and application thereof
  • NK trophoblast cell as well as preparation method and application thereof
  • NK trophoblast cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1 lentiviral vector construction

[0069] Whole gene synthesis tandem CD19 coding sequence and CD86 coding sequence KCD19CD86 (SEQ ID NO: 11), IL-21-CD8α coding sequence KIL21CD8α (SEQ ID NO: 8), CD64 coding sequence KCD64 (SEQ ID NO: 9), CD137L Coding sequence (SEQ ID NO:10), and restriction site BamHI / EcoRI or BamHI / NotI are added at both ends;

[0070] 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 HLU digested with the same restriction endonuclease ;

[0071] Table 1

[0072] 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)

[0073] The digested products were detected by agarose gel electrophoresis, the electrophoresis condition was 120V constant current electro...

Embodiment 2

[0074] Example 2 Recombinant lentivirus packaging and titer detection

[0075] 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:

[0076] (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, reseeding in T175 culture flask or d15 culture dish, 37°C, 5% CO 2 , cultivated under saturated humidity;

[0077] (2) After 24 hours, replace the cell cultu...

Embodiment 3

[0088] Construction and identification of embodiment 3 NK trophoblast cells

[0089] 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;

[0090] The prepared NK trophoblast cells were subjected to flow cytometric detection, such as figure 2 As shown, the expression rate of K562 to CD19 was 99.32%, the expression rate to CD86 was 99.99%, the expression rate to IL21 was 98.29%, the expression rate to CD64 was 99.18%, and the expression rate to CD137L (41BBL) was 99.56%. %, indicating that NK trophoblast cells were successfully constructed.

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Abstract

The invention provides an NK trophoblast cell as well as a preparation method and application thereof. The NK trophoblast cell expresses membrane proteins CD19, CD86, IL21, CD64 and CD137L; the IL-21 forms an IL-21-CD8[alpha] fusion protein through the IL-21 and a CD8[alpha] trans-membrane region, and the IL-21-CD8[alpha] fusion protein is expressed on the surface of the NK trophoblast cells; the CD86 comprises an amino acid sequence as shown in SEQ ID NO: 1; and the IL-21-CD8[alpha] 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 cell is synergistically activated by the factors, so that the effect of inducing the NK cell to differentiate and mature in vitro is realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to NK trophoblast cells and a preparation method and application thereof. Background technique [0002] In recent years, cellular immunotherapy has shown great potential in cancer treatment and is considered to be the most promising method to overcome cancer. Among them, T cell-based immunotherapy technology has received extensive attention and is a current research hotspot. However, the major histocompatibility complex (MHC) greatly limits the scope of application of T cell immunotherapy. Unlike MHC-restricted T cells, NK cells do not require HLA matching, so they can be used as heterogeneous off-the-shelf cell drugs for immunotherapy of patients, and have important clinical application prospects. [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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K14/705C07K14/54C07K14/735C12N5/0783C12N15/867C12N7/01C12N15/24C12N15/12C12R1/93C12R1/91
CPCC07K14/70503C07K14/70532C07K14/54C07K14/70535C07K14/70575C12N15/86C12N7/00C12N5/0646C12N2510/00C12N2502/30C12N2740/15021C12N2740/15043
Inventor 赵礼军熊建民
Owner HENAN HUALONG BIOLOGICAL TECH
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