CN118895244A - A polypeptide for improving NK cell killing activity and its application - Google Patents

Abstract

The invention belongs to the technical field of life science, and in particular relates to a polypeptide for improving NK cell killing activity and application thereof. The polypeptides for improving the NK cell killing activity are LL-37 polypeptide and Nigroain-E1 polypeptide, the Nigroain-E1 polypeptide can inhibit the cytotoxicity of the LL-37 polypeptide after being co-cultured with NK cells, the biological activity of the LL-37 polypeptide is not influenced, and the LL-37 polypeptide can improve the killing activity of the NK cells to tumor cells in the presence of Nigroain-E1 polypeptide. After the two polypeptides are combined, the expression level of perforin in NK cells can be effectively promoted, so that the killing activity of the perforin on tumor cells is improved.

Description

A polypeptide for improving NK cell killing activity and its application

Technical Field

The present invention belongs to the field of life science technology, and more specifically, relates to a polypeptide for improving the killing activity of NK cells and an application thereof.

Background Art

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 are increasingly gaining attention for their role in anti-tumor and anti-viral treatment. Therefore, improving the killing activity of NK cells is of great significance for the treatment of tumors and viral infections.

NK cells, or Natural Killer Cells, are a type of lymphocyte with unique phenotypes and functions, and are an important component of the innate immune system. Compared with T cells and B cells, they have a major advantage in that they can quickly identify 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-based immunotherapy.

NK cell therapy has been widely used in autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. This is due to the unique immune response mechanism of NK cells, which can largely inhibit the over-activation of the immune system and improve the body's own immune function. In addition, the receptors on the surface of NK cells can recognize markers on the surface of target cells. With their own spectral antiviral ability, they 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, improving the killing activity of NK cells can enhance their potential in disease treatment.

There have been many studies devoted to improving the killing activity of NK cells, but existing methods still have limitations. For example, existing drugs or culture media that enhance the killing activity of NK cells have poor promotion effects and may bring side effects or the risk of immune overactivation to their clinical application. Its role is also limited by many factors.

Therefore, it is necessary to develop a polypeptide that enhances the killing activity of NK cells and give full play to the role of NK cells in disease treatment.

Summary of the invention

The first object of the present invention is to provide a polypeptide that improves the killing activity of NK cells.

The second purpose of the present invention is to provide the application of the above-mentioned polypeptide for improving the killing activity of NK cells. After the combined treatment of Nigroain-E1 polypeptide and LL-37 polypeptide, the killing activity of NK cells is improved and the expression level of perforin is significantly increased.

In order to achieve the first purpose of the present invention, the technical solution adopted by the present invention is:

A polypeptide for improving the killing activity of NK cells, wherein the polypeptide is LL-37 polypeptide and Nigroain-E1 polypeptide.

Preferably, the amino acid sequence of the LL-37 polypeptide is as 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 (SEQ ID NO: 1);

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-Ary-Ile-Cys-Arg-Asp (SEQ ID NO: 2).

In order to achieve the second purpose of the present invention, the technical solution adopted by the present invention is:

The use of the above polypeptide in the preparation of drugs for improving the killing activity of NK cells.

Preferably, the separated NK cells are inoculated into a culture medium supplemented with LL-37 polypeptide and Nigroain-E1 polypeptide for co-culture.

Preferably, the NK cells are NK cells derived from peripheral blood.

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.

Preferably, the culture medium is AIM-V medium.

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

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

Compared with the prior art, the beneficial effects of the present invention are mainly:

The present invention provides a polypeptide for improving the killing activity of NK cells and its application. Nigroain-E1 polypeptide can inhibit the cytotoxic activity of LL-37 polypeptide and does not affect the activity of NK cells. In the presence of Nigroain-E1 polypeptide, LL-37 polypeptide can enhance the killing activity of NK cells against tumor cells. Moreover, through Western blotting experiments, it is fully proved that the expression level of perforin in NK cells after co-culture of the above two polypeptides is significantly increased, thereby enhancing its killing activity against tumor cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a morphological diagram of NK cells cultured in Example 1;

FIG2 is a graph showing the results of NK cell killing activity detection after polypeptide treatment;

FIG3 shows the protein expression of perforin in NK cells after polypeptide treatment.

DETAILED DESCRIPTION

The technical scheme of the present invention is further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

Example 1

Acquisition of NK cells: Take human peripheral blood, add heparin anticoagulation, and dilute the peripheral blood with 2 volumes of DPBS solution. Then take a 50mL sterile centrifuge tube, add 15mL of Ficoll separation solution to each tube, and then slowly and evenly add 15mL of diluted peripheral blood to the centrifuge tube, centrifuge at 2500rpm for 15min, discard the upper plasma, carefully transfer the cells in the middle white cloudy layer to a new centrifuge tube, resuspend with 2 volumes of DPBS solution, centrifuge at 2500rpm for 15min, discard the supernatant, and the precipitate is the peripheral blood mononuclear cell (PBMC).

PBMCs 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 a 37°C, 5% CO ₂ saturated humidity incubator for induction 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 by the above induction culture. Fresh culture medium was supplemented every 3 days to maintain the cell density at 3×10 ⁶ cells/mL. NK cells were collected after 14 days of culture, and the growth of NK cells obtained by culture in treatment group 1 was observed under a microscope. The results are shown in Figure 1.

As shown in Figure 1, NK cells are round in shape, grow in clusters, and are relatively uniform and stable overall.

Example 2

Detection of cytotoxicity of LL-37 polypeptide and Nigroain-E1 polypeptide on peripheral blood NK cells: The NK cells cultured in Example 1 were washed twice with PBS buffer, digested with 0.25% trypsin for 3 minutes, and the digestion solution was discarded after terminating the digestion. The NK cells were resuspended with AIM-V culture medium (as shown in Table 1) corresponding to treatment groups 1-3 and control groups 1-2, respectively, and the density was adjusted to 1×10 ⁴ cells/mL, inoculated into 96-well plates, and cultured in groups as described in Table 1. At the same time, a positive control group was set: a cell well without the above polypeptide; a negative control group: a well containing only the above polypeptide but no cells; 3 duplicate wells were set for each group and cultured for 24 hours. 50 μL of MTT solution was added to each well, and the culture was continued for 4 hours. 150 μL of DMSO was added and mixed evenly, and the reaction was carried out for 10 minutes. Then, the absorbance value of each well at 492 nm was detected by an ELISA instrument, and the cell survival rate was calculated. The results are shown in Table 1. The calculation formula of NK cell survival rate is as follows: survival rate = (OD experimental group-OD negative control group)/(OD positive control group-OD negative control group)×100%.

Table 1

It shows that the cytotoxic activity of LL-37 polypeptide is inhibited in the presence of Nigroain-E1 polypeptide. It can be intuitively observed from the results in Table 1 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 need to add serum, and can inhibit the cytotoxicity of LL-37 polypeptide only under the action of Nigroain-E1 polypeptide.

Example 3

Detection of NK cell tumor cell killing activity of LL-37 polypeptide and Nigroain-E1 polypeptide: 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 culture medium described in Table 1 and cultured continuously for 24 hours at 37°C and 5% CO _2. At the same time, a control group 3 was set up: NK cells obtained in Example 1. NK cells were used as effector cells and their density was adjusted to 1×10 ⁵ cells/mL. K562 human chronic myeloid leukemia tumor cells in the logarithmic growth phase were used as target cells, and their density was adjusted to 5×10 ³ cells/mL. The above NK cell suspension and K562 cell suspension were inoculated into a 96-well plate in equal volumes (i.e., the effector-target ratio was 20:1). At the same time, single target cell wells and single effector cell wells were set as controls. Three replicate wells were set in each well. The inoculated 96-well plate was placed in a 37°C, 5% CO ₂ incubator for culture. After 12 hours, 10 μL of CCK-8 reagent was added to each well, and the incubation continued for 3 hours. The absorbance value (OD value) of each well at a wavelength of 450 nm was measured by an enzyme-linked immunosorbent assay, and the average value was obtained. The OD value was calculated according to the following formula: Killing rate (%) = [1-(OD value of the treatment group-OD value of the single effector cell)/(OD value of the single target cell)] × 100%. The results are shown in Figure 2.

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

Compared with control group 3, control group 1 was able to maintain the killing activity of NK cells after adding Nigroain-E1 peptide alone, while control group 2 increased the killing activity of NK cells after adding LL-37 peptide alone. When the effector-target ratio was 20:1, the tumor cell killing rate of treatment group 1 reached 82.35%, the killing rate of treatment group 2 was 80.02%, and the killing rate of treatment group 3 was 81.67%, indicating that the combined treatment of Nigroain-E1 peptide and LL-37 peptide can enhance the killing activity of NK cells against tumor cells.

Example 4

Western Blot experiment to detect the expression of perforin: NK cells cultured in Example 2 were collected, digested with 0.25% trypsin, and resuspended in the culture medium described in Table 1, that is, treatment 1-3 and control group 1-2 were set. In addition, the NK cells obtained by culturing for 14 days in Example 1 were used as control group 3, and a cell suspension with a density of 3×10 ⁶ cells/mL was prepared and inoculated into a 6-well plate. The plate was cultured at 37°C and 5% CO ₂ for 12 h, and the cell suspension was centrifuged at 5000 rpm for 10 min. The supernatant was discarded, the lower layer of cells was collected, the cells were washed twice with PBS, RIPA cell lysis buffer was added to fully lyse the cells, and then centrifuged in a centrifuge (4°C, 10000 rpm, 10 min), and the obtained supernatant was the total cell lysate.

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

Denaturation of protein: In order to fully denature the protein, heat it in a constant temperature water bath at 100°C for 5 minutes to obtain a protein sample, and then cool it to room temperature.

Electrophoresis: Slowly drop the above protein sample into the gel comb holes and apply current to separate the proteins until the dye migrates to the bottom of the gel.

Membrane transfer: Take the gel out of the electrophoresis tank, cut out filter paper and PVDF membrane of the same size according to the size of the gel, arrange the sandwich structure in the order of filter paper + gel + PVDF membrane, and transfer the membrane for 2 hours.

Blocking of membrane: After the transfer, place the membrane in an incubation box containing blocking solution (TBST solution containing 5% skim milk powder) and shake slowly on a shaker at room temperature for 1 hour. Wash the PVDF membrane with TBST buffer to thoroughly wash away the residual blocking solution.

Antibody binding: Add perforin and GAPDH primary antibodies to react with proteins on the membrane and incubate overnight at 4°C. On the second day, discard the primary antibody solution, wash the membrane 3 times with TBST solution, 5 minutes each time, then add the secondary antibody and incubate at room temperature for 1 hour. Discard the secondary antibody solution and wash the membrane 3 times with TBST solution again, 5 minutes each time.

Color development: Place the PVDF membrane in a chemiluminescent imager, add chemiluminescent solution so that it evenly covers the surface of the membrane, and record the results after development.

The results of the above protein blotting experiment are shown in Figure 3. It can be clearly observed that compared with the control group 3, the expression of perforin in the NK cells after treatment in the treatment groups 1-3 was significantly increased. Perforin is a key molecule for NK cells to exert their killing effect, and its protein expression is closely related to the killing activity of NK cells. It can be seen that the above results show that under the joint action of LL-37 polypeptide and Nigroain-E1 polypeptide, the expression 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 of perforin in NK cells treated only with Nigroain-E1 polypeptide or LL-37 polypeptide is lower than that in the treatment group 1.

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 above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims (9)

1. A polypeptide for improving the killing activity of NK cells, characterized in that the polypeptide is LL-37 polypeptide and Nigroain-E1 polypeptide. 2. A polypeptide for improving NK cell killing activity according to claim 1, characterized in that the amino acid sequence of the LL-37 polypeptide is as shown in SEQ ID NO: 1; the amino acid sequence of the Nigroain-E1 polypeptide is as shown in SEQ ID NO: 2. 3. Use of the polypeptide as claimed in claim 1 or 2 in the preparation of a drug for improving the killing activity of NK cells. 4. Use of the polypeptide according to claim 3 in the preparation of a drug for improving the killing activity of NK cells, characterized in that the separated NK cells are inoculated into a culture medium supplemented with LL-37 polypeptide and Nigroain-E1 polypeptide, and cultured together. 5. Use of the polypeptide according to claim 3 in the preparation of a drug for improving the killing activity of NK cells, characterized in that the NK cells are NK cells derived from peripheral blood. 6. Use of the polypeptide according to claim 3 in the preparation of a drug for improving the killing activity of NK cells, 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. 7. Use of the polypeptide according to claim 6 in the preparation of a drug for improving the killing activity of NK cells, characterized in that the culture medium is AIM-V culture medium. 8. Use of the polypeptide according to claim 3 in the preparation of a drug for improving the killing activity of NK cells, characterized in that the seeding density of the NK cells is 1-5×10 ⁴ cells/mL. 9. Use of the polypeptide according to claim 3 in the preparation of a drug for increasing the expression of perforin in NK cells.