CN114835779A

CN114835779A - Polypeptide and application thereof in promoting proliferation of human natural killer cells and preparing human natural killer cell culture medium

Info

Publication number: CN114835779A
Application number: CN202210658768.2A
Authority: CN
Inventors: 陈飞
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-06-13
Filing date: 2022-06-13
Publication date: 2022-08-02

Abstract

The invention discloses a polypeptide and application thereof in promoting proliferation of human natural killer cells and preparing a human natural killer cell culture medium. Natural killer cells are innate immune cells against tumors and viruses, play an important role in innate immunity, play an important role in early antitumor and immune surveillance, and can directly kill tumor cells without antigen-specific recognition. Because the number of natural killer cells in human body is small, large-scale research and experiment are limited. The polypeptide of the invention has the function of promoting the proliferation of the human natural killer cells, and can be used for developing and preparing a culture medium for promoting the in vitro proliferation of the human natural killer cells.

Description

Polypeptide and application thereof in promoting proliferation of human natural killer cells and preparing human natural killer cell culture medium

Technical Field

The invention belongs to the field of biochemistry, relates to polypeptide and natural killer cell culture, and particularly relates to polypeptide and application thereof in promoting human natural killer cell proliferation and preparing a human natural killer cell culture medium.

Background

In the early 70 s of the 20 th century, researchers discovered spontaneous cytotoxic effects among lymphocytes, and found a new lymphocyte subset, which was named Natural killer cells (NK). Natural killer cells are innate immune cells against tumors and viruses, play an important role in innate immunity, play an important role in early antitumor and immune surveillance, and can directly kill tumor cells without antigen-specific recognition. Because the number of natural killer cells in human body is small, large-scale research and experiment are limited. The research on the cell biological characteristics and the amplification in vitro to obtain a large amount of high-purity NK cells become hot problems.

Polypeptides are compounds of linked amino acids, linked by peptide bonds, and generally refer to compounds consisting of three or more amino acids. The polypeptide may include active polypeptide and artificially synthesized polypeptide, and the active polypeptide has certain physiological effect as protein hydrolysate. The artificially synthesized polypeptide can freely control the amino acid connection sequence according to the requirement, provides possibility for constructing a polypeptide library, and becomes an important source for activity screening.

The invention is particularly provided based on the discovery of polypeptides for promoting the proliferation of human natural killer cells.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a polypeptide and application thereof in promoting the proliferation of human natural killer cells and preparing a human natural killer cell culture medium.

The purpose of the invention is realized by the following technical scheme:

a polypeptide with amino acid sequence RNDWTMDEQ.

The application of the polypeptide in promoting the in vitro proliferation of the human natural killer cells.

The application of the polypeptide in preparing a culture medium for promoting the in vitro proliferation of the human natural killer cells.

Has the advantages that:

the polypeptide provided by the invention has the function of promoting the proliferation of human natural killer cells, and can be used for developing and preparing a culture medium for promoting the in vitro proliferation of the human natural killer cells.

Drawings

FIG. 1 is a liquid phase detection diagram of a polypeptide; retention time 8.273min, HPLC purity 95.327%.

FIG. 2 is a flow identification chart of NK cell isolation culture, which represents that the phenotype ratio of CD3-CD56+ of NK cells is obviously increased.

FIG. 3 shows the result of western blot detection, in which the expression of lethality-related proteins in NK cells of polypeptide J group is significantly increased.

Detailed Description

The following examples are only intended to illustrate the essence of the present invention, but not to limit the scope of the present invention.

First, experimental material

The polypeptide J with the Sequence of RNDWTMDEQ (Sequence No.1) is synthesized by Nanjing peptide cereal Biotech Co., Ltd by a conventional solid phase synthesis method. The molecular formula of the polypeptide J is C ₄₈ H ₇₁ N ₁₅ O ₁₉ S, molecular weight is 1194.23, molecular ion peak [ M + H ] is detected by mass spectrum] ⁺ 1195.2. HPLC purity 95.327%, see fig. 1.

NK cell culture medium, Sadet biopharmaceutical GmbH (mainly composed of RPMI 1640, IL-15, IL-2, and SCF, only used for in vitro amplification culture of NK cells separated from human lymphocytes, and having no functions of cell selection, induction, and differentiation); RPMI 1640 medium and fetal bovine serum, GIBCO.

MTT reagent, monoclonal antibody, Westernblot primary antibody and secondary antibody, and Biyunyan.

Second, Experimental methods

1. Isolated culture of NK cells

According to the operating specification of separating and culturing NK cells from peripheral blood, 100mL of peripheral anticoagulation blood of healthy volunteers is taken, mononuclear cells are separated, and the mononuclear cells are re-suspended and inoculated in a 6-well plate by using an NK cell culture medium at 37 ℃ and 5% CO ₂ Culturing under conditions, changing the culture medium half times every 2 days, and adjusting cell culture density to 5 × 10 ⁴ and/mL. NK cells cultured for 14d were collected for subsequent experiments. The proportion of CD3-CD56+ phenotype cells is detected by adding a proper amount of PerCP-Cy5.5 labeled CD3 and FITC labeled CD 56.

2. MTT method for detecting in-vitro proliferation activity of NK cells

The cultured NK cells of 14 days were digested, resuspended in RPMI 1640 complete medium (RPMI 1640 medium containing 10% fetal bovine serum), and resuspended at 5X 10 ³ Cells/well were plated in 96-well culture plates at 100. mu.L per well, and divided into a control group, a polypeptide J-L group and a polypeptide J-H group, each of 6 wells. After 24h of incubation, 100 μ L of fresh medium was added to each well to continue incubation: the control group was supplemented with fresh RPMI 1640 complete medium, the polypeptide J-L group with RPMI 1640 complete medium containing 25. mu.g/mL of polypeptide J, and the polypeptide J-H group with RPMI 1640 complete medium containing 50. mu.g/mL of polypeptide J. Culturing for 48 and 72h, respectively, taking out a culture plate, adding 20 μ L of 5m culture plate into each wellg/mLMTT solution, continuously culturing for 4h, removing the supernatant, adding 150 mu LDMSO into each hole, oscillating for 5min, and measuring the absorbance value at 490nm on an automatic microplate reader system.

3. Western blot method for detecting NK cell killing activity marker protein expression level

The cultured 14d NK cells were digested, resuspended in RPMI 1640 complete medium (RPMI 1640 medium containing 10% fetal calf serum) and suspended at 2.5X 10 ⁵ Cells/well were plated in 6-well plates, 1mL per well, and divided into control, polypeptide J-L and polypeptide J-H groups, 2 wells per group. After 24h of incubation, 1mL of fresh medium was added to each well to continue incubation: the control group was supplemented with fresh RPMI 1640 complete medium, the polypeptide J-L group with RPMI 1640 complete medium containing 25. mu.g/mL of polypeptide J, and the polypeptide J-H group with RPMI 1640 complete medium containing 50. mu.g/mL of polypeptide J. After culturing for 48 hours, the cells were collected, lysed, and total cell protein was extracted, protein concentration was measured by BCA method, and protein was separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel using GAPDH as an internal reference and protein loading was 30. mu.g. The membrane was flowed at constant pressure of 90V to transfer the gel protein to a Nitrocellulose (NC) membrane. The NC membrane is placed into phosphate Tween buffer (PBST) solution containing 5% skimmed milk powder and is sealed for 2h at room temperature. Diluted primary antibody solutions (Granzyme B, Perforin, β -actin) were then added separately and incubated overnight at 4 ℃. The membrane was washed 3 times with PBST solution for 10min each time, and diluted horseradish peroxidase-labeled secondary antibody was added and incubated for 2h at room temperature. PBST solution is used for washing the membrane for 3 times, ECL luminescent solution is added for color development, and then photographing and analysis are carried out.

4. Statistical analysis

Statistical analysis is carried out by using SPSS19.0 statistical software, single-factor analysis of variance (ANOVA) is used for comparing multiple groups of means, independent sample t test is used for comparing two groups of means, and the difference P <0.05 has significance.

Third, experimental results

1. NK cell isolation culture

The results of the measurement of the proportion of CD3-CD56+ phenotype cells are shown in FIG. 2, and the proportion of CD3-CD56+ phenotype rapidly increased from 14.9% to 78.5% of the initial culture. The CD3-CD56+ phenotype cell is the NK cell. The results of this assay indicate that NK cells have been successfully isolated from peripheral blood.

2. NK cell in vitro proliferative Activity

The absorbance values of each group are shown in table 1, the proliferation activity of NK cells of the polypeptide J-L group and the polypeptide J-H group is obviously stronger than that of a control group (the difference from the control group has statistical significance, # P <0.05), and the proliferation activity of NK cells is stronger when the concentration of the polypeptide J is higher (the difference from the polypeptide J-L group has statistical significance, # P < 0.05).

TABLE 1 Absorbance values for each group

3. Expression level of NK cell killing activity marker protein

The Granzyme B and Perforin proteins are 2 key proteins for the NK cells to exert killing activity, and the expression level determines the strength of the killing activity of the NK cells. Western blot detection results are shown in FIG. 3, expression levels of the proteins Granzyme B and Perforin in NK cells of the polypeptide J-L group and the polypeptide J-H group are obviously higher than those of a control group, and the higher the concentration of the polypeptide J is, the higher the expression level of the 2 proteins is.

The results show that the polypeptide provided by the invention has the effect of promoting the proliferation of human natural killer cells, can also improve the killing activity of NK cells, and can be used for developing and preparing culture media of human natural killer cells.

Sequence listing

<110> Chenfei

<120> a polypeptide and application thereof in promoting proliferation of human natural killer cells and preparing human natural killer cell culture medium

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Arg Asn Asp Trp Thr Met Asp Glu Gln

1 5

Claims

1. A polypeptide, characterized by: the amino acid sequence is RNDWTMDEQ.

2. Use of the polypeptide of claim 1 for promoting proliferation of human natural killer cells in vitro.

3. Use of the polypeptide of claim 1 for preparing a medium for promoting proliferation of human natural killer cells in vitro.