阅读说明：本技术 一种多肽及用于nk细胞培养、制备nk细胞培养基的用途 (Polypeptide and application of polypeptide in NK cell culture and preparation of NK cell culture medium ) 是由 于海涛 杨晓旭 于 2020-06-30 设计创作，主要内容包括：本发明公开了一种多肽及用于NK细胞培养、制备NK细胞培养基的用途,该多肽的氨基酸序列如下：KGNP(X<Sub>X</Sub>)TDHQVFWMWMLGEAAYD；其中,X<Sub>X</Sub>代表D或G。本发明提供的多肽既可以促进NK细胞体外增殖,又可以提高NK细胞的杀伤力,因而可以用于促进NK细胞体外增殖并增强其杀瘤活性,并用于制备可以促进NK细胞体外增殖并增强其杀瘤活性的细胞培养基。(The invention discloses a polypeptide and application thereof in NK cell culture and preparation of an NK cell culture medium, wherein the amino acid sequence of the polypeptide is as follows: KGNP (X) X ) TDHQVFWMWMLGEAAYD, respectively; wherein, X X Represents D or G. The polypeptide provided by the invention can promote the in vitro proliferation of the NK cells and improve the killing power of the NK cells, so that the polypeptide can be used for promoting the in vitro proliferation of the NK cells and enhancing the tumor killing activity of the NK cells and preparing a cell culture medium capable of promoting the in vitro proliferation of the NK cells and enhancing the tumor killing activity of the NK cells.)

1. A polypeptide having the amino acid sequence:

KGNP(X_X)TDHQVFWMWMLGEAAYD；

wherein, X_XRepresents D or G.

2. The polypeptide of claim 1, wherein: it is a polypeptide whose N and C termini are not chemically modified.

3. The polypeptide of claim 1, wherein: it is a polypeptide whose N and C termini are chemically modified.

4. The polypeptide of claim 3, wherein the N-terminus is modified by acetylation and the C-terminus is modified by amidation in the form of:

Ac-KGNP(X_X)TDHQVFWMWMLGEAAYD-NH₂；

wherein, X_XRepresents D or G.

5. Use of the polypeptide of any one of claims 1 to 4 for promoting NK cell proliferation in vitro.

6. Use of the polypeptide of any one of claims 1 to 4 for enhancing NK cell lethality.

7. Use of the polypeptide of any one of claims 1 to 4 for the preparation of an in vitro culture medium for NK cells, which can promote the in vitro proliferation and enhance the killing of NK cells.

Technical Field

The invention relates to the field of polypeptide and tumor immune cells, in particular to polypeptide and application thereof in NK cell culture and preparation of an NK cell culture medium.

Background

Normally, the immune system can recognize and eliminate tumor cells in the tumor microenvironment, but for survival and growth, the tumor cells can adopt different strategies, so that the immune system of the human body is inhibited and the tumor cells cannot be normally killed, thereby surviving in each stage of the anti-tumor immune response. The above-described feature of tumor cells is called immune escape, and in order to better understand the multi-stage, multi-stage complexity of tumor immunity, the concept of tumor-immune cycle has been developed and proposed. The tumor-immune cycle is divided into the following seven links: 1. tumor antigen release; 2. tumor antigen presentation; 3. priming and activating effector T cells; 4. migration of T cells to tumor tissue; 5. tumor tissue T cell infiltration; 6. t cells recognize tumor cells; 7. eliminating tumor cells. Abnormalities in any of these links can lead to failure of the anti-tumor-immune cycle and immune escape. Different tumors can inhibit the effective recognition and killing of tumor cells by the immune system through the abnormality of different links, thereby generating immune tolerance and even promoting the occurrence and development of tumors.

Tumor immunotherapy is a therapeutic method for controlling and eliminating tumors by restarting and maintaining tumor-immune circulation and restoring normal anti-tumor immune response of the body. Including monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy, small molecule inhibitors, and the like. In recent years, the tumor immunotherapy is well-known, and has now demonstrated strong antitumor activity in the treatment of solid tumors such as melanoma, non-small cell lung cancer, renal cancer and prostate cancer, and many tumor immunotherapy drugs have been approved by FDA in the united states for clinical application. Because of its excellent curative effect and innovativeness, immunotherapy for tumors is judged by the journal of science in 2013 as the most important scientific breakthrough every year.

Cellular immunotherapy is a tumor immunotherapy with immune cells, and NK and CIK immune cells are used for research. NK cells are important immune cells of the body, are not only related to tumor resistance, virus infection resistance and immune regulation, but also participate in hypersensitivity reaction and autoimmune disease occurrence under certain conditions, and can recognize target cells and kill mediators.

Cellular immunotherapy has high requirements for the input of immune cells such as NK, and therefore, it is necessary to obtain a sufficient number of NK cells in vitro before infusion. Therefore, how to promote the proliferation of NK cells and improve the lethality of NK cells in vitro becomes an important research direction in the field of cellular immunotherapy.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings in the prior art and provide a polypeptide and application thereof in NK cell culture and preparation of an NK cell culture medium.

The technical scheme for realizing the purpose is as follows:

a polypeptide having the amino acid sequence as follows:

KGNP(X_X)TDHQVFWMWMLGEAAYD；

wherein, X_XRepresents D or G.

Preferably, it is a polypeptide whose N and C termini are not chemically modified.

Preferably, it is a polypeptide whose N and C termini are chemically modified.

More preferably, the N-terminus is modified by acetylation and the C-terminus is modified by amidation, in the form:

Ac-KGNP(X_X)TDHQVFWMWMLGEAAYD-NH₂；

wherein, X_XRepresents D or G.

The polypeptide is used for promoting the in vitro proliferation of NK cells.

The use of the above polypeptide for enhancing NK cell lethality.

The polypeptide is used for preparing an NK cell in-vitro culture medium, and the culture medium can promote the in-vitro proliferation of NK cells and enhance the killing property of the NK cells.

Has the advantages that:

the polypeptide provided by the invention can promote the in vitro proliferation of the NK cells and improve the killing power of the NK cells, so that the polypeptide can be used for promoting the in vitro proliferation of the NK cells and enhancing the tumor killing activity of the NK cells and preparing a cell culture medium capable of promoting the in vitro proliferation of the NK cells and enhancing the tumor killing activity of the NK cells.

Drawings

FIG. 1 shows the result of Western Blot assay; compared with a control group, the contents of the polypeptide group granzyme B and the perforin at different concentrations are obviously increased, and the higher the concentration is, the more obvious the increase is.

Detailed Description

First, test materials

And (2) entrusting a polypeptide to be tested to a wrapping mechanism for synthesis by using a solid-phase synthesis method, adopting a Fmoc (Fmoc) N-terminal protection strategy, sequentially connecting corresponding amino acids according to a resin solid-phase synthesis method, sequentially removing Fmoc-protection groups during the process, cutting the peptide to obtain a crude product, and separating and purifying by using column chromatography to obtain the polypeptide. The purity is not lower than 98%. Structure confirmation was performed by HPLC and MS.

The sequences of the polypeptides to be tested, Sequence No.1 to Sequence No.4, are as follows, the N-and C-termini being chemically unmodified:

KGNPPTDHQVFWMWMLGEAAYD(Sequence NO.1)；

KGNPPTDHQVFWMGMLGEAAYD(Sequence NO.2)；

KGNPDTDHQVFWMWMLGEAAYD(Sequence NO.3)；

KGNPGTDHQVFWMWMLGEAAYD(Sequence NO.4)。

the human lymphocyte separation medium was purchased from Tianjin grade Marine Biotechnology, Inc.

Recombinant human interleukin 2(rhIL-2) was purchased from Byunnan.

NK cell culture medium was purchased from CellGenix, Germany.

MTT and DMSO were purchased from Sigma.

Granzyme a, granzyme B, perforin-resistant were purchased from Invitrogen, usa.

Second, test method

1. Peripheral blood NK cell culture and identification

Collecting peripheral blood of healthy human 20mL, anticoagulating with heparin, diluting with 20mL PBS buffer solution, slowly adding lymphocyte separation solution, centrifuging at 4 deg.C for 20min at 650g, collecting white cell layer, washing with normal saline, adding washed cells into NK cell culture medium containing IL-2 500U/mL, adjusting density to 2.5 × 10⁶Per L, 5% CO at 37 ℃₂Culturing, adding NK cell culture medium containing 500U/mL IL-2 every 2-3 days and adjusting cell density to 2.5 × 10⁶And L. After 12 days of culture, NK cells were labeled with PerCP-cy5.5-CD3 and FITC-CD56 antibodies for phenotypic detection by flow cytometry.

2. Method for measuring NK cell proliferation activity by MTT method

Collecting cultured NK cells for 12 days, making into cell suspension, inoculating into 96-well plate at density of 5000 cells and 200 μ L per well, and culturing at 37 deg.C with 5% CO₂Conditioned for 24h and the medium was replaced with NK cell medium containing 4, 8. mu.M of the polypeptide to be tested and a control without the addition of the polypeptide. Each provided with 6 multiple wells. At 37 deg.C, 5% CO₂The culture was continued for 48 hours and the cells were removed, 20. mu.L of MTT solution (5mg/mL) was added to each well,after 4h of culture, the supernatant was discarded, 150. mu.L of DMSO was added to each well, the crystals were sufficiently dissolved by gentle shaking, and the absorbance (OD) of each well was measured at 570nm, and the cell proliferation rate of the different polypeptide groups was calculated by the following formula, based on the cell proliferation rate of the control group being 100%:

proliferation rate (%) ═ OD polypeptide group/OD control group × 100%.

3. Western Blot method for measuring killing power of NK cells

Collecting cultured NK cells of 12d, and making into 2 × 10⁵The cell suspension was seeded in 6-well plates at 2 mL/well in 37 ℃ with 5% CO₂Conditioned for 24h and the medium was replaced with NK cell medium containing 4, 8. mu.M of the polypeptide to be tested and a control without the addition of the polypeptide. At 37 deg.C, 5% CO₂Culturing for 48h, collecting cells, washing with PBS, lysing the lysate, determining the protein concentration, performing SDS-PAGE electrophoresis on each group by 30 μ g of protein, transferring the membrane, sealing with 5% skimmed milk powder, adding granzyme B, perforin and GAPDH primary antibody, incubating overnight at 4 ℃, washing the membrane, adding horseradish peroxidase-labeled secondary antibody, incubating for 2h at room temperature, developing, and performing gray scanning analysis.

4. Data processing

The variance analysis is carried out by adopting SPSS 17.0 software, the data are expressed by mean value plus or minus standard deviation, the single-factor variance analysis is adopted among groups, and the difference with P less than 0.05 has statistical significance.

Third, test results

1. Peripheral blood NK cell culture and identification

The NK cell is a CD3^-CD56⁺Phenotypic cells, which account for only about 5% of mononuclear cells isolated from peripheral blood. In the results of the phenotypic assay by flow cytometry after 12d in culture, CD3^-CD56⁺The phenotype is improved to (40.55 +/-3.96)%, which indicates that the NK cells are successfully induced after the peripheral blood mononuclear cells are cultured by the NK cell culture medium containing the IL-2.

2. Effect of Polypeptides on NK cell proliferation Activity

MTT test results As shown in Table 1, the OD of the polypeptide group was obtained at different concentrations compared with the control group₅₇₀All values are obviously increasedAnd the higher the concentration, the more obvious the increase. It can be seen that the polypeptides of Sequence No.1 to Sequence No.4 can effectively promote the proliferation of NK cells.

TABLE 1 OD values and proliferation rates of polypeptide groups

3. Effect of Polypeptides on NK cell lethality

The Western Blot test result is shown in FIG. 1, compared with the control group, the contents of the granzyme B and the perforin in the polypeptide group with different concentrations are obviously increased, and the increase is more obvious when the concentration is higher. Granzyme B and perforin are two important substances that determine NK cell lethality: the former is the most important serine protease in NK cell granules, can enter target cells and activate Caspase cascade reaction, thereby rapidly causing DNA (deoxyribonucleic acid) breakage of the target cells and causing rapid apoptosis; the latter is a cytotoxic granule present in the cytoplasm of NK cells, which when NK comes into contact with target cells can release perforin, forming a polyperpin tubular channel in the target cell membrane, resulting in lysis of the target cells. Therefore, the polypeptides from Sequence No.1 to Sequence No.4 can effectively improve the killing power of NK cells.

In conclusion, the polypeptide provided by the invention can promote the in vitro proliferation of the NK cells and improve the killing power of the NK cells, so that the polypeptide can be used for promoting the in vitro proliferation of the NK cells and enhancing the tumor killing activity of the NK cells and preparing a cell culture medium capable of promoting the in vitro proliferation of the NK cells and enhancing the tumor killing activity of the NK cells.

Sequence listing

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Gly Glu Ala Ala Tyr Asp

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