A polypeptide for enhancing the killing activity of NK cells and its application

Abstract

The present invention belongs to the field of life science and technology. More specifically, it relates to a polypeptide for enhancing the killing activity of NK cells and its application. The polypeptides for enhancing the killing activity of NK cells in the present invention are LL-37 polypeptide and Nigroain-E1 polypeptide. After co-culturing the above polypeptides with NK cells, Nigroain-E1 polypeptide can inhibit the cytotoxicity of LL-37 polypeptide and does not affect the biological activity of LL-37 polypeptide. In the presence of Nigroain-E1 polypeptide, LL-37 polypeptide can enhance the killing activity of NK cells against tumor cells. After treatment with the combination of the above two polypeptides, it can effectively promote the expression level of perforin in NK cells, thereby enhancing its killing activity against tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of life science technology, and more specifically, relates to a polypeptide for enhancing the killing activity of NK cells and its application. Background Art

[0002] In recent years, with the rapid development of biotechnology, scientists have gained a deeper understanding of the immune system. As a type of immune cell with strong killing activity, NK cells have increasingly attracted attention in anti-tumor and anti-viral therapies. Therefore, enhancing the killing activity of NK cells is of great significance for the treatment of tumors and viral infections.

[0003] NK cells, namely natural killer cells, are a type of lymphocyte with unique phenotypes and functions and are an important part of the innate immune system. Compared with T cells and B cells, they have a major advantage that they can quickly recognize and kill severely damaged cells or tumor cells without antigen stimulation, and play an important role in early defense, maintaining the body's immune balance, resisting diseases, and cell immunotherapy.

[0004] NK cell therapy has been widely used in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. By virtue of the unique immune response mechanism of NK cells, it can largely inhibit the overactivation of the immune system and improve the body's own immune function. In addition, the receptors on the surface of NK cells can recognize the markers on the surface of target cells. With its broad-spectrum antiviral ability, it can quickly kill virus-infected or abnormal cells and release cytokines to interfere with the proliferation of viruses, thereby more effectively killing viruses and tumor cells. It can be seen that NK cells have achieved good therapeutic effects in the treatment of various diseases. Therefore, enhancing the killing activity of NK cells can enhance its potential in disease treatment.

[0005] Currently, many studies have been devoted to enhancing the killing activity of NK cells, but the existing methods still have limitations. For example, the existing drugs or culture media for enhancing the killing activity of NK cells have poor promotion effects, and may bring side effects or risks of immune overactivation in their clinical applications. Their effects are also limited by various factors.

[0006] Therefore, it is necessary to develop a polypeptide for enhancing the killing activity of NK cells to give full play to the role of NK cells in disease treatment. Summary of the Invention

[0007] The first object of the present invention is to provide a polypeptide for enhancing the killing activity of NK cells.

[0008] The second object of the present invention is to provide the application of the above-mentioned polypeptide for enhancing the killing activity of NK cells. After the combined treatment with Nigroain-E1 polypeptide and LL-37 polypeptide, the killing activity of NK cells is enhanced, and the expression level of perforin is significantly increased.

[0009] In order to achieve the first object of the present invention, the technical solution adopted by the present invention is:

[0010] A polypeptide for enhancing the killing activity of NK cells, wherein the polypeptide is LL-37 polypeptide and Nigroain-E1 polypeptide.

[0011] Preferably, the amino acid sequence of the LL-37 polypeptide is as shown in SEQ ID NO: 1;

[0012] Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser-Lys-Glu-Lys-Ile-Gly-Lys-Glu-Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-Asn-Leu-Val-Pro-Arg-Thr-Glu-Ser (SEQ ID NO: 1);

[0013] The amino acid sequence of the Nigroain-E1 polypeptide is as shown in SEQ ID NO: 2;

[0014] Asp-Cys-Thr-Arg-Trp-Ile-Ile-Gly-Ile-Asn-Gly-Arg-Ile-Cys-Arg-Asp (SEQ ID NO: 2).

[0015] In order to achieve the second object of the present invention, the technical solution adopted by the present invention is:

[0016] The application of the above polypeptide in the preparation of a drug for enhancing the killing activity of NK cells.

[0017] Preferably, the isolated NK cells are inoculated into a culture medium supplemented with LL-37 polypeptide and Nigroain-E1 polypeptide and co-cultured.

[0018] Preferably, the NK cells are NK cells derived from peripheral blood.

[0019] Preferably, the final concentration of the LL-37 polypeptide in the culture medium is 10 - 30 μg / mL, and the final concentration of the Nigroain-E1 polypeptide in the culture medium is 25 - 75 μg / mL.

[0020] Preferably, the culture medium is AIM-V culture medium.

[0021] Preferably, the seeding density of the NK cells is 1-5×10 4 cells / mL.

[0022] In addition, the present invention provides the use of the polypeptide for enhancing the killing activity of NK cells in the preparation of a drug for increasing the expression level of perforin in NK cells.

[0023] Compared with the prior art, the beneficial effects of the present invention mainly lie in:

[0024] The present invention provides a polypeptide for enhancing the killing activity of NK cells and its application. The Nigroain-E1 polypeptide can inhibit the cytotoxic activity of the LL-37 polypeptide and does not affect the activity of NK cells. In the presence of the Nigroain-E1 polypeptide, the LL-37 polypeptide can enhance the killing activity of NK cells against tumor cells. Moreover, through Western blot experiments, it is fully demonstrated that the expression level of perforin in NK cells co-cultured with the above two polypeptides is significantly increased, thereby enhancing their killing activity against tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Morphological diagram of NK cells cultured in Example 1;

[0026] Figure 2 Detection result diagram of the killing activity of NK cells after polypeptide treatment;

[0027] Figure 3 Protein expression level of perforin in NK cells after polypeptide treatment. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The following further describes the technical solutions of the present invention in conjunction with specific embodiments. However, those skilled in the art should understand that the following examples are only used to illustrate the present invention and should not be regarded as a limitation of the present invention. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0029] Example 1

[0030] Isolation of NK cells: Heparin anticoagulant was added to human peripheral blood, and the peripheral blood was diluted with DPBS solution at a volume ratio of 2:1. Then, 50 mL sterile centrifuge tubes were taken, and 15 mL Ficoll separation solution was added to each tube. Subsequently, 15 mL diluted peripheral blood was slowly and evenly added to the centrifuge tubes, and centrifuged at 2500 rpm for 15 min. After aspirating and discarding the upper plasma layer, the cells in the middle white cloudy layer were carefully aspirated and transferred to a new centrifuge tube, resuspended with DPBS solution at a volume ratio of 2:1, and centrifuged at 2500 rpm for 15 min. The supernatant was discarded, and the precipitate was peripheral blood mononuclear cells (PBMC).

[0031] The PBMC were inoculated into AIM-V medium containing 55 ng / mL IL-21 and 800 IU / mL IL-2 at a cell density of 3×10 6 cells / mL, and placed in an incubator at 37 °C with 5% CO2 and saturated humidity for induced culture. After 5 days of culture, AIM-V medium containing 800 IU / mL IL-2 and 12 ng / mL IL-18 was added to the cell suspension obtained from the above induced culture. Fresh medium was supplemented every 3 days to keep the cell density at 3×10 6 cells / mL. After 14 days of culture, NK cells were collected, and the growth of the NK cells obtained from Treatment Group 1 was observed under a microscope. The results are shown in Figure 1 .

[0032] It can be seen from Figure 1 that the morphology of NK cells is round, growing in aggregates, and is relatively uniform and stable as a whole.

[0033] Example 2

[0034] Cytotoxicity detection of LL-37 polypeptide and Nigroain-E1 polypeptide on peripheral blood NK cells: The NK cells obtained from the culture in Example 1 were washed twice with PBS buffer, digested with 0.25% trypsin for 3 min. After terminating the digestion, the digestion solution was poured out, and the NK cells were resuspended with the corresponding AIM-V medium of Treatment Groups 1-3 and Control Groups 1-2 (as shown in Table 1) respectively, and the density was adjusted to 1×10 4Cells were inoculated into 96-well plates at a density of [X] cells / mL and cultured according to the groups described in Table 1. At the same time, a positive control group was set up: cell wells without the above-mentioned polypeptide; a negative control group: wells containing only the above-mentioned polypeptide without cells; each group had 3 replicate wells and was cultured for 24 h. 50 μL of MTT solution was added to each well, and the culture was continued for 4 h. Then, 150 μL of DMSO was added and mixed evenly, and the reaction was carried out for 10 min. Then, the absorbance values of each well at 492 nm were measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated. The results are shown in Table 1. The calculation formula for the survival rate of NK cells is as follows: Survival rate = (ODexperimental group - ODnegative control group) / (ODpositive control group - ODnegative control group) × 100%.

[0035] Table 1

[0036]

[0037]

[0038] It shows that the cytotoxic activity of LL-37 polypeptide is inhibited in the presence of Nigroain-E1 polypeptide. From the results in Table 1, it can be intuitively observed that Nigroain-E1 polypeptide can effectively inhibit the cytotoxic effect of LL-37 polypeptide. Compared with the traditional method of inhibiting the cytotoxic activity of LL-37 polypeptide, the present invention does not require the addition of serum and can inhibit the cytotoxicity of LL-37 polypeptide only under the action of Nigroain-E1 polypeptide.

[0039] Example 3

[0040] Detection of the killing activity of LL-37 polypeptide and Nigroain-E1 polypeptide on NK cells against tumor cells: The NK cells cultured in Example 1 were collected, digested with 0.25% trypsin, and then resuspended to prepare a cell suspension with a density of 1×10 6 cells / mL. The cells were inoculated into the medium described in Table 1 and continuously cultured at 37 °C and 5% CO2 for 24 h. At the same time, control group 3 was set up: the NK cells obtained in Example 1. The NK cells were used as effector cells and adjusted to a density of 1×10 5 cells / mL. The K562 human chronic myelogenous leukemia tumor cells in the logarithmic growth phase were used as target cells and adjusted to a density of 5×10 3cells / mL, inoculate the above NK cell suspension and K562 cell suspension in equal volumes into a 96-well plate (i.e., the effector-to-target ratio is 20:1). At the same time, set up wells with only target cells and wells with only effector cells as controls, with 3 replicates in each well. After inoculation, place the 96-well plate in an incubator at 37°C and 5% CO2 for culture. After 12 h, add 10 μL of CCK-8 reagent to each well and continue to incubate for 3 h. Use an enzyme-linked immunosorbent assay detector to measure the absorbance value (OD value) of each well at a wavelength of 450 nm, calculate the average value, and calculate the OD value according to the following formula: Killing rate (%) = [1 - (OD value of the treatment group - OD value of the pure effector cells) / (OD value of the pure target cells)] × 100%. The results are as Figure 2 shown.

[0041] It can be seen from Figure 2 the results that under the action of Cathelicidin LL-37 polypeptide and Nigroain-E1 polypeptide, the killing activity of NK cells against K562 cells is significantly improved.

[0042] Compared with control group 3, after adding Nigroain-E1 polypeptide alone in control group 1, the killing activity of NK cells can be maintained, while after adding LL-37 polypeptide alone in control group 2, the killing activity of NK cells increases. When the effector-to-target ratio is 20:1, the tumor cell killing rate of treatment group 1 reaches 82.35%, the killing rate of treatment group 2 is 80.02%, and the killing rate of treatment group 3 is 81.67%, indicating that the combined treatment of Nigroain-E1 polypeptide and LL-37 polypeptide can improve the killing activity of NK cells against tumor cells.

[0043] Example 4

[0044] Western Blot experiment to detect the expression level of perforin: Collect the NK cells obtained from the culture in Example 2, digest them with 0.25% trypsin, and resuspend the NK cells according to the medium described in Table 1, that is, set up treatment groups 1-3 and control groups 1-2. In addition, use the NK cells obtained from the culture in Example 1 for 14 days as control group 3, and prepare a cell suspension with a density of 3×10 6 cells / mL, inoculate it into a 6-well plate, and culture it at 37°C and 5% CO2 for 12 h. Centrifuge the cell suspension at 5000 rpm for 10 min, discard the supernatant, collect the cells in the lower layer, wash the cells 2 times with PBS, add RIPA cell lysate to fully lyse the cells, and then centrifuge in a centrifuge (4°C, 10000 rpm, 10 min). The obtained supernatant is the total cell lysate.

[0045] Determination of protein concentration: The BCA quantification method was used to calculate the protein concentration in the above total cell lysate. According to the measured protein results above, the protein concentrations of each group of samples were adjusted to be the same. According to the volume ratio of protein to protein loading buffer of 1:4, the corresponding volume of protein loading buffer was added thereto.

[0046] Protein denaturation: To fully denature the protein, it was heated in a constant temperature water bath at 100 °C for 5 min to obtain the protein sample, which was then cooled to room temperature.

[0047] Electrophoresis: The above protein sample was slowly dropped into the gel comb holes, an electric current was applied to separate the proteins until the dye migrated to the bottom of the gel.

[0048] Membrane transfer: The gel was taken out of the electrophoresis tank, filter paper and PVDF membrane of the same size were cut according to the size of the gel, and a sandwich structure was arranged in the order of filter paper + gel + PVDF membrane, and transferred for 2 h.

[0049] Membrane blocking: After the membrane transfer was completed, the membrane was placed in an incubation box containing a blocking solution (TBST solution containing 5% skim milk powder) and slowly shaken at room temperature on a shaker for 1 h. The PVDF membrane was washed with TBST buffer to thoroughly wash away the remaining blocking solution.

[0050] Antibody binding: Perforin and GAPDH primary antibodies were added to react with the proteins on the membrane and incubated overnight at 4 °C. The next day, the primary antibody solution was discarded, the membrane was washed 3 times with TBST solution for 5 min each time, then the secondary antibody was added and incubated at room temperature for 1 h. The secondary antibody solution was discarded, and the membrane was washed again 3 times with TBST solution for 5 min each time.

[0051] Color development: The PVDF membrane was placed in a chemiluminescence imager, and chemiluminescence solution was added to evenly cover the surface of the membrane, and the results were recorded after development.

[0052] The results of the above Western blot experiment are as Figure 3 shown. It can be clearly observed that compared with control group 3, the expression level of perforin in NK cells after treatment with treatment groups 1-3 was significantly increased. Perforin is a key molecule for NK cells to exert their killing effect, and its protein expression level is closely related to the killing activity of NK cells. It can be seen that the above results show that under the combined action of LL-37 polypeptide and Nigroain-E1 polypeptide, the expression level of perforin in NK cells is significantly increased, which can effectively enhance the killing activity of NK cells against tumor cells, thereby more effectively clearing pathogens or tumor cells, while the protein expression level of perforin in NK cells treated with only Nigroain-E1 polypeptide or LL-37 polypeptide is lower than that of treatment group 1.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described in the above with specific implementation schemes. On the basis of the present invention, some modifications or replacements can be made, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection required by the present invention.

Claims

1. A polypeptide composition for improving NK cell killing activity, characterized in that: The polypeptide composition is a polypeptide composition consisting of LL-37 polypeptide and Nigroain-E1 polypeptide. The amino acid sequence of the LL-37 polypeptide is shown in SEQ ID NO: 1: Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser-Lys-Glu-Lys-Ile-Gly-Lys-Glu-Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-Asn-Leu-Val-Pro-Arg-Thr-Glu-Ser; The amino acid sequence of the Nigroain-E1 polypeptide is shown in SEQ ID NO: 2: Asp-Cys-Thr-Arg-Trp-Ile-Ile-Gly-Ile-Asn-Gly-Arg-Ile-Cys-Arg-Asp.

2. Use of the polypeptide composition as claimed in claim 1 in preparing a culture medium for improving the killing activity of NK cells.

3. The use according to claim 2, characterized in that: The separated NK cells were inoculated into a culture medium supplemented with LL-37 polypeptide and Nigroain-E1 polypeptide for co-culture.

4. The use according to claim 2, characterized in that: The NK cells are NK cells derived from peripheral blood.

5. The use according to claim 2, characterized in that: The final concentration of the LL-37 polypeptide in the culture medium is 10-35 μg / mL, and the final concentration of the Nigroain-E1 polypeptide in the culture medium is 25-75 μg / mL.

6. The use according to claim 5, characterized in that: The culture medium is AIM-V culture medium.

7. The use according to claim 3, characterized in that: The seeding density of the NK cells is 1-5×10 4 Pieces / mL.

8. Use of the polypeptide composition according to claim 1 in preparing a culture medium for increasing the expression of perforin in NK cells.

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