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Polypeptide capable of reinforcing killing activity of NK (natural killer) cells and application of polypeptide

A technology of NK cells and killing activity, applied in the field of life sciences, can solve the problems of inability to mass-produce, clinically uncertain hazards cannot be assessed, and increase costs, etc., to achieve the effect of enhancing killing activity, promoting apoptosis, and increasing expression

Active Publication Date: 2020-06-19
XINLU CELL BIOTECHNOLOGY (HAINAN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the above methods often require the combined application of multiple cytokines to achieve the purpose of inducing and expanding NK cells, which not only increases the cost, but also cannot be assessed for clinically uncertain hazards, and cannot be mass-produced industrially.

Method used

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  • Polypeptide capable of reinforcing killing activity of NK (natural killer) cells and application of polypeptide
  • Polypeptide capable of reinforcing killing activity of NK (natural killer) cells and application of polypeptide
  • Polypeptide capable of reinforcing killing activity of NK (natural killer) cells and application of polypeptide

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Experimental program
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Effect test

Embodiment 1

[0036] Peptide Design and Synthesis

[0037] The polypeptide of the present invention is derived from gMYL6, combined with the conservative type of gMYL6, truncated and designed to obtain a small molecular polypeptide comprising 15 amino acids in total, and the sequence is shown in SEQ ID NO.1. The polypeptide of the present invention is synthesized by solid-phase synthesis. For the specific synthesis method, refer to the reference Recent Reports of Solid-Phase Cyclohexapeptide Synthesis and Applications. Molecules. 2018 Jun; 23(6): 1475, and the final product is identified by ESI-MS method. The purified peptide mass spectrum is shown in figure 1 . Mass spectrometry analysis showed that the molecular weight of the purified polypeptide was 1789Da, which was consistent with the calculated value.

Embodiment 2

[0039] Culture and phenotype identification of NK cells derived from human umbilical cord blood

[0040] 1) NK cell activation

[0041] Activating and culturing umbilical cord blood NK cells: using lymphocyte medium to adjust the density of umbilical cord blood mononuclear cells to 1-5×10 6 / mL, then add 1mL activated culture factor and 1000IU / mL recombinant human interleukin-2, at 37℃, 5%CO 2 Cultured in a saturated humidity environment for 3 days, and the cell fluid was collected.

[0042] 2) NK cell proliferation

[0043] Cells were obtained from the activated cell liquid, and the cell density was adjusted to 1-5×10 with X-VIVO 15 cell culture medium (LONZA, USA). 6 / mL, then add 1000IU / mL recombinant human interleukin-2, at 37°C, 5% CO 2 Cultured in a saturated humidity environment, supplemented with fresh medium every 3 days and adjusted the cell density to 1-5×10 6 / mL, cultivated for 25 days, and harvested cord blood NK cells.

[0044] 3) Phenotype identification ...

Embodiment 3

[0049] CCK-8 Assay to Detect the Killing Activity of NK Cells on Tumor Cells

[0050] Take the NK cells cultured for 20 days, culture them with the culture medium containing 0 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL of the polypeptide of the present invention respectively for 8 hours, wash thoroughly with PBS 3 times to remove the polypeptide, and prepare 6×10 4 / mL cell suspension, as effector cells; K562 human chronic myelogenous leukemia cells in the logarithmic growth phase were made into 3×10 4 / mL cell suspension, as the target cells. The effector cell suspension and the target cell suspension were inoculated into 96-well culture plates in equal volumes (immediate effect-to-target ratio 2:1), and a single effector cell well, a single target cell well and a blank well were set up at the same time, with 5 replicate wells in each group. At 37°C, 5% CO 2 Incubate in the incubator for 8 hours; add 10 μL of CCK-8 reagent, incubate for 4 hours, measure the absorbance (OD) of ...

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Abstract

The invention provides polypeptide capable of reinforcing killing activity of NK (natural killer) cells and an application of the polypeptide, and belongs to the technical field of life science. The amino acid sequence of the polypeptide capable of reinforcing killing activity of NK (natural killer) cells is as shown in SEQID No.1. After the polypeptide and the NK cells are incubated, the killingactivity of the NK cells to tumor cells can be notably reinforced, and the expression quantity of perforin and CD107a of the NK cells is increased.

Description

technical field [0001] The invention belongs to the technical field of life sciences, and in particular relates to a polypeptide capable of enhancing NK cell killing activity and its application. Background technique [0002] The incidence of malignant tumors is increasing year by year, and has become one of the diseases with a high mortality rate in the world. It was once regarded as an incurable disease. Among them, tumor recurrence and metastasis are one of the root causes of malignant tumors that are difficult to cure. The root of it lies in the immune escape of tumor cells. Natural killer (NK) cells are a member of innate immune cells and play an extremely important role in the immune surveillance of tumors. NK cells are the body's first line of defense against tumors and infections. In the process of killing tumor cells, they can directly exert their killing effect without the recognition of tumor-specific antigens. Therefore, NK cells are more effective in killing t...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N5/0783A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/4716C12N5/0646C12N2501/998
Inventor 杨兆勇姬钰滢金媛媛陈静邹森樊帅王忠博石北辰孙正阳
Owner XINLU CELL BIOTECHNOLOGY (HAINAN) CO LTD
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