A method for improving NK cell function

Abstract

The present invention belongs to the fields of biotechnology and pharmaceutical technology, and in particular to a method for improving NK cell function. The method of the present invention is based on the overexpression of deubiquitinase USP30 technology, and the overexpression of deubiquitinase USP30 technology includes the construction of deubiquitinase USP30 overexpression plasmid pCDH-USP30, and the construction of NK-92MI cell line NK92-pCDH-USP30 overexpressing USP30 protein. The method of the present invention can effectively improve the effector function of NK cells, including restoring mitochondrial function and promoting the production of effector molecules IFN-γ and TNF-α, and increasing the killing effect on stably transfected HBV virus HepG2 cells, i.e., HepG2.2.15 cells. It has an important enlightenment effect on the exhaustion of NK cell function under various pathological conditions, and has broad application prospects in the treatment of HBV chronic infection, tumors and other fields.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and medicine technology, and in particular relates to a method for improving the function of NK cells. Background Art

[0002] NK cells are the main cells of the natural immune system, essential for innate immunity and specific immunity, and play an important immune function in immune regulation, hematopoiesis, anti-infection and anti-tumor through their cytotoxic activity and secreted lymphokines. Unlike cytotoxic T cells, they do not need prior contact with antigens to mediate effector functions, and NK cell-mediated cytotoxicity and cytokine release can affect the activity of other innate immune cells (dendritic cells, macrophages and neutrophils) and give NK cells regulatory functions.

[0003] After identifying tumor cells, NK cells kill tumor cells through multiple pathways, such as releasing the killing mediators perforin and granzyme to cause target cell apoptosis, expressing membrane TNF family molecules to induce target cell apoptosis, and antibody-dependent cytotoxicity. However, due to the physical barriers of solid tumors, as well as the inhibitory immune microenvironment such as hypoxia, nutrient competition, and inhibitory cytokines, the number and quality of NK cells in tumor patients decrease, and the activation of NK cells decreases, making NK cells in a state of disability. At the same time, there is tumor immune escape, so the anti-tumor function of NK cells in the body cannot be fully exerted. Coincidentally, during chronic viral or bacterial infection, long-term antigen stimulation causes severe damage to the effector function of NK cells, which is manifested by increased expression of inhibitory receptors and significantly reduced secretion of effector molecules IFN-γ and TNF-α.

[0004] Mitochondria are double-membrane organelles that are involved in metabolism and energy supply. They are always in a state of highly dynamic balance between fission and fusion. Mitochondrial fusion, fission, biogenesis and mitochondrial autophagy determine the morphology, quality and abundance of mitochondria and are strictly controlled to ensure that mitochondrial function adapts to the energy and metabolic needs of cells. In addition to providing energy for cells, mitochondria play an important role in amino acid and lipid metabolism, cellular oxidative stress, cell signal transduction and apoptosis. Cells produce byproducts during mitochondrial metabolism, including ROS, Ca 2+Etc. When viral infection or inflammation, hypoxia, and nutritional stress occur in the tumor microenvironment, the mitochondrial respiratory chain will be affected, resulting in increased electron generation, and a large number of electrons combined with oxygen, leading to excessive ROS production in the mitochondria. In addition to the production of ROS in the respiratory chain, excessive mitochondrial fragmentation will also increase the production of ROS. ROS in mitochondria are transferred to the cytoplasm, and can also undergo lipid peroxidation with intracellular lipids to cause cell ferroptosis. ROS-mediated mitochondrial damage will affect the electron transport chain to further reduce ATP production, and may cause energy depletion of cells, further affecting cell function. External factors such as changes in nutrients in the living environment of some cells lead to metabolic reprogramming. Continuous antigen stimulation and hypoxia in the tumor microenvironment can also lead to increased ROS in immune cells, inducing mitochondrial function exhaustion and oxidative damage, further accelerating tumor progression. In most cases, NK cells are in a relatively quiescent state, usually metabolizing glucose into pyruvate, acetyl-CoA, etc. or performing fatty acid oxidation (FAO) in mitochondria through the TCA cycle to obtain energy. Mitochondrial metabolism is a key factor in maintaining the vitality and function of immune cells. When stimulated by antigens, NK cells undergo metabolic reprogramming to obtain a large amount of energy and metabolic intermediates to meet their biosynthesis, thereby performing rapid proliferation, differentiation, and enhancing effector functions. However, during viral infection or long-term antigen stimulation such as tumors, mitochondrial metabolic disorders occur, and immune cells show phenomena such as increased mitochondrial ROS levels and hyperpolarization and reduced mitochondrial mass, leading to mitochondrial dysfunction. The above changes will further affect the function of immune cells, resulting in a weakened antiviral response. The secretion of cytotoxic cytokines by NK cells with mitochondrial dysfunction is significantly reduced, resulting in reduced anti-tumor and anti-viral capabilities. However, studies on NK cell mitochondrial dysfunction caused by chronic HBV infection have not been reported.

[0005] Mitophagy is a process in which autophagosomes selectively engulf mitochondria and are subsequently degraded by lysosomes. Its function is to selectively remove damaged mitochondria and is also an important process for maintaining their abundance and health. Mitophagy can be divided into two categories based on the different autophagosome recognition pathways: (1) ubiquitin-related mitophagy, which is mediated by the PINK1 (PTEN Induced Kinase 1) / PRKN (Parkin RBR E3 Ubiquitin Protein Ligase) pathway; (2) ubiquitin-independent mitophagy, which is mediated by mitophagy receptor proteins. The number of mitochondria will be adjusted according to changes in metabolic needs, and the PINK1-Parkin pathway plays an important role in this process. USP30, as a member of the deubiquitinating enzyme family, is mainly located in the outer membrane of mitochondria and is a cysteine ​​protease. Earlier studies have shown that USP30 is involved in the regulation of intracellular mitochondrial morphology. Subsequent studies have found that USP30 can reverse mitophagy driven by PARK2 and PINK1. PARK2 acts as a ubiquitin ligase to mark damaged mitochondria for clearance, while USP30 acts as a deubiquitinating enzyme that removes ubiquitin tags and inhibits mitochondrial autophagy.

[0006] In a liver cancer mouse model with a high-fat diet, high expression of USP30 in its tumor cells can promote the deubiquitination of ATP citrate lyase (ACLY) and fatty acid synthase (FASN), thereby promoting lipogenesis and tumor development. In addition, mitochondrial dysfunction is also associated with a large number of physiological diseases, including neurodegenerative diseases, cancer, cardiovascular diseases, and metabolic disorders. Most of the existing research on USP30 is centered on Parkinson's disease-related treatments. Currently, companies such as Mission Therapeutics and Mitobridge have developed USP30 inhibitors. Now studies have found that USP30 can also regulate the killing function of CTL. In addition, as a deubiquitinating enzyme on the outer membrane of mitochondria, USP30-mediated deubiquitination inhibits mitochondrial autophagy, clears damaged mitochondria, and maintains mitochondrial quality. However, USP30 is extremely complex in different disease models and different cells. The mechanism of USP30's involvement in NK cell mitochondrial dysfunction is still unclear, and there are no reports on targeting USP30 to play a role in NK cell antiviral infection.

[0007] The mitochondrial regulatory mechanism of NK cells is extremely complex. Targeting mitochondria to enhance the effector function of NK cells is the key to anti-infection and anti-tumor treatment based on NK cells. Summary of the invention

[0008] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a method for improving the function of NK cells, which is based on the technology of overexpressing the deubiquitinating enzyme USP30. The technology of overexpressing the deubiquitinating enzyme USP30 includes the construction of a deubiquitinating enzyme USP30 overexpression plasmid pCDH-USP30 and the construction of a NK-92MI cell line NK92-pCDH-USP30 overexpressing USP30 protein.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] One of the purposes of the present invention is to provide a method for improving the phenotypic stability of NK cells and enhancing the immunomodulatory function, the method comprising increasing the expression of human mitochondrial deubiquitinase USP30. Preferably, the encoding nucleic acid of the deubiquitinase USP30 comprises SEQ ID NO: 1, its degenerate sequence or its spliceosome nucleotide sequence; the deubiquitinase USP30 protein comprises SEQ ID NO: 2, its spliceosome or its variant amino acid sequence.

[0011] Preferably, the product for expressing the deubiquitinating enzyme USP30 protein is selected from a combination of one or more of an overexpression plasmid, a protein agonist and a compound agonist; the method for expressing the deubiquitinating enzyme USP30 protein is preferably to transfect NK cells by a lentiviral packaging method.

[0012] The second purpose of the present invention is to provide a deubiquitinase USP30 overexpression plasmid pCDH-USP30, wherein the overexpression plasmid pCDH-USP30 is a recombinant plasmid obtained by connecting the target gene USP30 to the pCDH-CMV-MCS-EF1-copGFP vector.

[0013] The third object of the present invention is to provide a method for constructing the above-mentioned deubiquitinase USP30 overexpression plasmid pCDH-USP30, which is characterized by comprising the following steps:

[0014] (1) Primer synthesis: the forward primer sequence is 5'-GC TCT AGA ATG CTG AGC TCC CGG GCC GA-3', and the reverse primer sequence is 5'-CG GGA TCC TCA TTC TTC AGA CTT GCA CT-3';

[0015] (2) PCR amplification of USP30 gene: Using NK92-MI cDNA as a template, PCR amplification was performed using the forward primer and reverse primer described in step (1) to obtain the target gene;

[0016] (3) Construction of USP30 overexpression plasmid: The target gene obtained in step (2) and the pCDH-CMV-MCS-EF1-copGFP vector were digested, purified and ligated with restriction endonucleases Xba I and BamH I, and the ligation product was transformed into Escherichia coli to obtain the pCDH-USP30 overexpression plasmid.

[0017] Preferably, the PCR amplification system in step (2) is: 2×ApexHF FS PCR Master Mix 25 μL, template 1 μL, 10 μM forward primer 2.5 μL, 10 μM reverse primer 2.5 μL, ddH 2 O 19μL, the total system is 50μL; the PCR amplification conditions are: 95℃ pre-denaturation for 5min, then 98℃10s, 60℃5s, 72℃2min for 35 cycles, and finally 72℃ extension for 2min.

[0018] Preferably, the enzyme digestion reaction system in step (3) comprises: 1 μL Xba I, 1 μL BamH I, 5 μL 10×Buffer, 2.5 μL vector plasmid or 20 μL PCR gel recovery product, supplemented with ddH 2 O until the total system is 50 μL.

[0019] Preferably, the ligation reaction system described in step (3) comprises: calculating the ratio of the PCR product recovered after enzyme digestion and the pCDH vector by vector mass × 1000 / vector length × 650: PCR product mass × 1000 / PCR product length × 650 = 1:3, taking the corresponding volume, 1 μL T4 DNA ligase, and supplementing with ddH 2 O until the total system is 10 μL.

[0020] The fourth object of the present invention is to provide a method for constructing a NK-92MI cell line NK92-pCDH-USP30 that overexpresses USP30 protein, comprising the following steps:

[0021] S1. Lentivirus packaging:

[0022] Transfecting the lentiviral packaging plasmids RRE, REV, VSVG and the overexpression plasmid pCDH-USP30 into human embryonic kidney epithelial cells HEK293T by calcium phosphate transfection method for lentiviral packaging to obtain packaged lentivirus;

[0023] S2. Construction of overexpression cell lines:

[0024] After the packaged lentivirus obtained in S1 is concentrated, the virus solution is infected with the NK-92MI cell line to obtain the NK-92MI cell line NK92-pCDH-USP30 overexpressing USP30. Preferably, in step S1, the amounts of lentiviral packaging plasmids RRE, REV, VSVG and recombinant plasmid pCDH-USP30 are 4.1 μg, 2.1 μg, 3.1 μg and 4.1 μg respectively.

[0025] The present invention also provides a NK-92MI cell line NK92-pCDH-USP30 overexpressing USP30 protein constructed by the above method.

[0026] The present invention also provides applications of the NK-92MI cell line NK92-pCDH-USP30 that overexpresses USP30 protein, including the following applications:

[0027] Application in increasing mitochondrial mass and mitochondrial membrane potential of NK92-MI cells, and increasing protein translation in mitochondria of NK92-MI cells;

[0028] Application in increasing the secretion of cytokines IFN-γ and TNF-α by NK92-MI cells;

[0029] Application in increasing the killing efficiency and antiviral effect of HepG2 cells stably transfected with HBV virus, namely HepG2.2.15 cells;

[0030] Application in reversing NK cell exhaustion in chronic viral infection and tumor microenvironment.

[0031] The chronic viral infection includes chronic HBV infection, chronic HCV infection, and HIV infection; the tumor includes leukemia, multiple myeloma, malignant lymphoma, liver cancer, lung cancer, and melanoma.

[0032] The NK-92MI cell line NK92-pCDH-USP30 overexpressing the USP30 protein can be used alone or in combination with other drugs.

[0033] Beneficial Effects

[0034] The present invention discloses a method for improving the function of NK cells. Compared with the prior art, the beneficial effects achieved by the present invention mainly include but are not limited to the following aspects:

[0035] (1) The present invention systematically compares the relationship between the expression level of human mitochondrial deubiquitinase USP30 and the function of NK cells derived from CHB, and verifies it using clinical cases of CHB, proving that USP30 is expressed at a low level in PBMC-derived NK cells of CHB patients. USP30 can be used as a new molecular marker and drug target for the diagnosis and treatment of chronic HBV infection patients (CHB). (2) The modified NK92-pCDH-USP30 cells of the present invention significantly increase the mitochondrial mass and membrane potential of NK92-MI cells, increase protein translation in the mitochondria of NK92-MI cells, and further enhance the ability of NK92-MI cells to exert their killing function.

[0036] (3) The modified NK92-pCDH-USP30 cells of the present invention significantly increased the secretion of NK92-MI cell cytotoxic cytokines IFN-γ and TNF-α, further enhanced the antiviral activity of the modified NK92-MI cells, and the killing effect on HepG2 cells stably transfected with HBV virus, namely HepG2.2.15 cells, and reduced the RNA level of HBV ccc DNA in HepG2 2.15 cells, so that the modified NK92-MI cell therapy technology can be applied to the treatment of chronic viral infections.

[0037] (3) The modified NK92-pCDH-USP30 cells of the present invention can reverse the state of NK cell exhaustion in chronic viral infection and tumor microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 :Gene sequencing results of NK cells in the liver of healthy mice and HBV-carrier mice;

[0039] Figure 2 :Confocal fluorescence microscopy was used to observe the expression of USP30 in NK cells from healthy volunteers (HD) and patients with chronic HBV infection (CHB);

[0040] Figure 3 :Flow cytometry analysis of the expression of USP30 in NK cells derived from HD and CHB;

[0041] Figure 4 :Flow cytometry analysis of mitochondrial membrane potential and mitochondrial mass of HD and CHB-derived NK cells;

[0042] Figure 5 :The relationship between the expression level of USP30 molecule detected by flow cytometry and the production level of IFN-γ and TNF-α of NK cells.

[0043] Figure 6: q-PCR and flow cytometry verification of the construction of the USP30-overexpressing NK92-MI cell line, namely NK92-pCDH-USP30 cells;

[0044] Figure 7 :Flow cytometry detection of mitochondrial membrane potential of NK92-pCDH-USP30 cells;

[0045] Figure 8 :Flow cytometry detection of IFN-γ and TNF-α production in NK92-pCDH-USP30 cells;

[0046] Fig. 9 :Flow cytometry detection of NK92-pCDH-USP30 cells killing HepG2 2.15 cells;

[0047] Fig.10 :Flow cytometry was used to detect the apoptosis level of HepG22.15 cells after incubation of HepG2 2.15 cells with NK92-pCDH-USP30 cell supernatant;

[0048] Fig.11 :q-PCR verified the expression of HBVcccDNA in HepG2 2.15 cells after incubation with NK92-pCDH-USP30 cell supernatant;

[0049] Fig.12 :Flow cytometry was used to detect the apoptosis level of NK92-pCDH-USP30 cells after incubation of NK92-pCDH-USP30 cells with blank culture medium and supernatant of HepG2 2.15, HepG2, and Huh7 tumor cells;

[0050] Fig.13 : Flow cytometry detection of the proportion of apoptosis of NK92-pCDH-USP30 cells after incubation of NK92-pCDH-USP30 cells with blank culture medium and supernatant of HepG2 2.15, HepG2, and Huh7 tumor cells. DETAILED DESCRIPTION

[0051] Hereinafter, the present invention will be described in detail. Before describing, it should be understood that the terms used in this specification and the appended claims should not be interpreted as being limited to the general meaning and dictionary meaning, but should be interpreted according to the meaning and concept corresponding to the technical aspects of the present invention on the basis of the principle that the inventor is allowed to appropriately define the terms for the best interpretation. Therefore, the descriptions presented here are only preferred examples for illustrative purposes and are not intended to limit the scope of the present invention, so that it should be understood that other equivalents or improvements can be obtained therefrom without departing from the spirit and scope of the present invention.

[0052] Definition of terms

[0053] As used herein, the term "NK cell" refers to natural killer cells, which are a type of immune cell that can participate in immune effects by secreting various cytokines.

[0054] As used herein, the term "IFN-γ" is interferon gamma, which is an important cytokine that can perform immunomodulatory functions. It was discovered for its antiviral activity. IFN-γ plays a key role in host defense through antiviral, antiproliferative and immunomodulatory functions. It can reflect immune cell effector functions.

[0055] As used herein, the term "TNF-α" refers to tumor necrosis factor α, which participates in normal inflammatory and immune responses and can coordinate the homeostasis of tissues by coordinating the production of other cytokines, cell survival and death. It is cytotoxic to various tumor cells and is an important factor in mediating the immune response to viral infection. It can reflect the effector function of immune cells.

[0056] The present invention is further described in detail below in conjunction with the accompanying drawings and specific experiments. Unless otherwise specified, the reagents, instruments, equipment and methods used in this patent are conventional commercial reagents, instruments, equipment and methods in the art. The following examples are only listed as examples of embodiments of the present invention and do not constitute any limitation to the present invention. It can be understood by those skilled in the art that modifications within the scope of the essence and concept of the present invention fall within the protection scope of the present invention.

[0057] Example 1

[0058] Expression of human mitochondrial deubiquitinase USP30 in NK cells derived from CHB patients 1.1 Materials

[0059] 1.1.1 Clinical samples for experiments

[0060] Blood samples from healthy volunteers and chronic hepatitis B patients were obtained from Qilu Hospital of Shandong University, and the experimental protocols were approved by the Scientific Research Ethics Committee of Qilu Hospital of Shandong University (approval number KYLL-2021(KS)-1034).

[0061] The clinical blood samples used in this experiment were divided into a healthy volunteer group (HD) and a chronic hepatitis B patient group (CHB). There was no statistical difference in gender ratio and age between the two groups of samples.

[0062] 1.1.2 Experimental animals

[0063] C57BL / 6J mice, male, 5 weeks old, were purchased from Huafukang Company (Beijing). Mice were kept in the animal room of this laboratory according to the SPF level requirements, in accordance with the animal husbandry and management regulations of Shandong University. All animal experiments were in accordance with the ethical requirements of Shandong University experimental animals and the animal protection guidelines of the National Institutes of Health (NIH). The experimental protocol was approved by the Ethics Committee of Qilu Medical College of Shandong University (approval number NO.21061).

[0064] 1.2 Methods

[0065] 1.2.1 Bioinformatics analysis of the expression of human mitochondrial deubiquitinase (USP30) in CHB-derived NK cells To analyze the expression of deubiquitinase-related genes in NK cells, we first sorted liver NK cells from 6 healthy mice and HBV-carrier mice, extracted RNA using the Trizol method, and analyzed the differentially expressed mitochondrial deubiquitinase-related genes in mouse liver NK cells after RNA-seq sequencing.

[0066] Gene sequencing results of NK cells in the liver of healthy mice and HBV-carrier mice are as follows Figure 1 As shown, Figure 1 The figure shows the expression of differentially expressed genes related to mitochondrial deubiquitinase in liver NK cells between healthy mice and HBV-carrier mice. The expression of USP30 in liver NK cells in HBV-carrier mice was significantly downregulated.

[0067] 1.2.2 Flow cytometry detection of mitochondrial activity and USP30 expression level of NK cells from CHB patients PBMCs were separated from fresh heparinized blood using the Ficoll-Hypaque density gradient method. 7 / mL) were labeled with human CD3-Percp-Cy5.5 (Biolegend) and CD56-APC-Cy7 (Biolegend) flow cytometry antibodies, and labeled in a 4°C refrigerator in the dark for 1 hour, shaking evenly every 30 minutes. After the labeling is completed, 1×PBS was added to wash away the excess antibodies; 200μL 1% paraformaldehyde was added to fix the cells, and the cells were labeled in a 4°C refrigerator in the dark for 30 minutes. After the fixation is completed, 1×PBS was added to wash away the fixative; 100μL cell permeabilization solution was continued to permeabilize the cells, and the cells were labeled in a 4°C refrigerator in the dark for 30 minutes. After completion, 1×PBS was added to wash away the fixative; or 200μL 0.2% Triton solution was added to permeabilize the cells.

[0068] USP30 labeling: After fixed, permeabilized and blocked cells, add 1 / 1000 dilution of recombinant Anti-USP30 antibody (Abclonal) for labeling at 4°C for 2h, add goat serum for blocking, and use goat anti-rabbit IgG-FITC (Abclonal) at a dilution of 1 / 1000 as secondary antibody. After washing, use confocal microscope LSM 900 or flow cytometer FACS Celesta (BD) for fluorescence photography or flow cytometry detection. The test results are shown in Figure 2 As shown, from Figure 2 It can be seen that compared with the control group, the USP30 fluorescence intensity of CHB-derived NK cells was significantly weakened.

[0069] Flow cytometry was used to detect the expression of USP30 in NK cells derived from HD and CHB. Figure 3 As shown, from Figure 3 It can be seen from the flow cytometry results that the positive percentage and average fluorescence intensity of the fluorescent secondary antibody were significantly reduced.

[0070] Detection of cell mitochondrial indicators: After the cell external labeling is completed, 100 μL 1×PBS (100 μL) is added to each tube, and 0.1 μL Mito-Tracker Green (50 μM) or Mito-Tracker Red CMXRos (50 μM) is added to each tube. Labeling is carried out at 37°C in the dark for 30 minutes, and shaking is performed every 10 minutes. After labeling is completed, 2-3 ml 1×PBS is added to each tube, centrifuged at 400g for 5 minutes, the supernatant is discarded, and 100 μL 1×PBS is added to each tube. Detection is performed by flow cytometry within 2 hours after staining.

[0071] Flow cytometry was used to detect the mitochondrial membrane potential and mitochondrial mass of HD and CHB-derived NK cells. Figure 4 As shown, from Figure 4 It can be seen that the mean fluorescence intensity of mitochondrial mass and membrane potential of CHB-derived NK cells was significantly reduced compared with the control group.

[0072] NK cell cytokine production detection: prepare cell activation medium (100 IU / mL IL-2 + 1 μg / mL ionomycin + 50 ng / mL PMA + 10 μg / mL brefeldin A), each 1×10 6 PBMC cells were added with 1 ml of activation medium and cultured in a 24-well plate for 4 hours. After labeling with CD3-Percp-Cy5.5 (Biolegend) and CD56-APC-Cy7 (Biolegend), they were fixed, permeabilized and blocked, and then labeled with IFN-γ-PE (Biolegend) and TNF-α-PE-CF594 (Biolegend), washed and tested. The results were analyzed by flow cytometry. Figure 5 As shown, the results showed that the levels of IFN-γ and TNF-α secreted by NK cells with low USP30 expression were significantly reduced compared with those of NK cells with high USP30 expression.

[0073] 1.2.3 Confocal microscopy analysis of USP30 expression levels in NK cells from CHB patients In order to more clearly observe the changes in USP30 expression in NK cells, we used the Ficoll-Hypaque density gradient method to separate PBMCs from fresh heparinized blood and then used the magnetic bead negative sorting method to sort primary NK cells from fresh PBMCs.

[0074] USP30 fluorescence confocal observation experiment: Add 100 μL of anti-fluorescence quenching sealing solution (containing DAPI) to the NK cells labeled with the secondary antibody above and incubate at room temperature for 3 minutes. Pipette 40 μL and drop it onto the slide, seal it with a cover glass, and use a confocal microscope Zeiss LSM 900 to take pictures. Figure 2 It can be seen that compared with the control group, the fluorescence intensity of the fluorescent secondary antibody linked to the USP30 antibody on CHB-derived NK cells was significantly weakened.

[0075] Example 2

[0076] 2.1 Materials

[0077] 2.1.1 Plasmids and cells

[0078] The expression system is a vector expressing human USP30; the vector is a lentiviral vector pCDH; the lentiviral plasmid packaging system for constructing the NK92-MI cell line overexpressing USP30 includes lentiviral packaging plasmids RRE, REV, VSVG and lentiviral expression vector pCDH-USP30. The cell lines required for the experiment are 293T cells and NK92-MI cells, all of which are preserved in this laboratory.

[0079] 2.1.2 Reagents

[0080] The high-fidelity enzyme (2×Easy Taq PCR Super Mix) used in PCR was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the gel purification kit was purchased from Omega Corporation in the United States; the restriction endonuclease was a product of New England Biolabs; T4 DNA ligase, nucleic acid standard molecular weight Marker, plasmid transfection reagent LipofectamineTM 2000, Trizol and protein standard molecular weight Marker were products of Thermo Scientific Company in the United States; the competent Escherichia coli DH5α was preserved in this laboratory.

[0081] 2.2 Methods

[0082] 2.2.1 Construction and identification of USP30 overexpression plasmid Primer synthesis: The human USP30 CDS region sequence was queried in the PubMed database. Based on this sequence, DNAMAN software was used to design the amplification primers for human USP30. The forward primer sequence was 5'-GC TCT AGA ATG CTG AGC TCC CGG GCC GA-3' (SEQ ID No: 3, the solid line part can be recognized by Xba I); the reverse primer sequence is 5'-CG GGA TCC TCA TTC TTC AGA CTT GCA CT-3' (SEQ ID No: 4, the solid line part can be recognized by BamH I). The primers were sent to Beijing Liuhe BGI Technology Co., Ltd. for synthesis.

[0083] The target gene USP30 was connected to the pCDH-CMV-MCS-EF1-copGFP vector to obtain the recombinant plasmid pCDH-USP30.

[0084] PCR amplification of USP30 gene: NK92-MI cDNA (SEQ ID No: 1 of GenBank accession number NC_84749) was used as template and amplified using the above primers. The amplification system was: 2×ApexHF FS PCR Master Mix 25 μL, template 1 μL, forward primer (10 μM) 2.5 μL, reverse primer (10 μM) 2.5 μL, ddH 2 O 19μL, the total system is 50μL.

[0085] The amplification conditions were: 95°C for 5 min pre-denaturation, then 98°C for 10 s denaturation, 60°C for 5 s annealing, 72°C for 2 min extension, 35 cycles of amplification, and finally 72°C for 2 min enhanced extension. The amplified product was detected by 1% agarose gel electrophoresis, and the gel was cut and recovered after the band position was confirmed.

[0086] Construction of USP30 overexpression plasmid: The lentiviral vector pCDH-CMV-MCS-EF1-copGFP and the product recovered by gel cutting after PCR amplification were double-digested with Xba I and BamH I restriction endonucleases. The enzyme digestion reaction system includes 1μL Xba I, 1μL BamH I, 5μL 10×Buffer, 2.5μL vector plasmid or 20μL PCR gel cutting product, supplemented with ddH 2 O to a total system of 50 μL, placed in a 37°C constant temperature water bath for 2 hours of enzyme digestion. 1% agarose gel electrophoresis, and gel cutting to recover the digestion product, the digestion of the PCR product and pCDH vector after digestion was calculated as vector mass × 1000 / vector length × 650: PCR product mass × 1000 / PCR product length × 650 = 1:3, take the corresponding volume, 1 μL T4 DNA ligase, supplement with ddH 2 O until the total system is 10 μL, mix well, and connect at 16°C for 12 hours. Transform the ligation product into competent E. coli DH5α, pick a single clone for amplification and extract the plasmid.

[0087] Identification of USP30 overexpression plasmid: The extracted plasmid DNA was amplified by PCR and digested with Xba I and BamH I, and the plasmid was sent to Beijing Liuhe BGI Gene Technology Co., Ltd. for sequencing. The sequencing results were compared with the USP30 gene sequence queried in PubMed. After the alignment was correct, it was named pCDH-USP30 (OV-USP30).

[0088] 2.2.2 Construction and identification of NK-92MI cell line overexpressing USP30 Lentiviral packaging: The lentiviral packaging plasmids RRE, REV, VSVG and recombinant plasmid pCDH-USP30 were transfected into human embryonic kidney epithelial cells HEK293T by calcium phosphate transfection for lentiviral packaging; the dosages of lentiviral packaging plasmids RRE, REV, VSVG and recombinant plasmid pCDH-USP30 were 4.1 μg, 2.1 μg, 3.1 μg and 4.1 μg, respectively.

[0089] Construction of overexpression cell line: After the packaged lentivirus was concentrated, the collected logarithmic growth phase NK-92MI cells (10 5 -10 6The infection was terminated by replacing the culture medium 4-6 hours after infection. NK-92MI cells infected with the control pCDH and USP30 virus solutions were collected 48 hours later.

[0090] Verification of NK-92MI cell lines overexpressing USP30: RNA was extracted from infected NK-92MI cells using the Trizol method or labeled with USP30 antibodies, and the expression of USP30 in NK-92MI cells after infection was verified by qPCR and flow cytometry. The results are as follows Figure 6 As shown, from Figure 6 The analysis showed that compared with the control group, the RNA expression level of USP30 was significantly upregulated by about 400 times, and the average fluorescence intensity of USP30 was also significantly increased.

[0091] Verification of the activity of the NK-92MI cell line overexpressing USP30: Flow cytometry was used to detect the mitochondrial activity and cytokine secretion ability of NK92-pCDH-USP30 (OV-USP30) cells. The results are as follows Figure 7-8 As shown. Figure 7 The results showed that compared with the control group, the mitochondrial membrane potential of NK92-pCDH-USP30 (OV-USP30) cells increased significantly. Figure 8 The results of the analysis showed that compared with the control group, the secretion levels of cytokines IFN-γ and TNF-α of NK92-pCDH-USP30 (OV-USP30) cells also increased significantly by about 20%, and the mean fluorescence intensity was also significantly enhanced.

[0092] Verification of the killing function of NK-92MI cell line overexpressing USP30: dilute the 5 mM CFSE stock solution with PBS at a ratio of 1:50, take 50 μL and add it to 1 mL of 1×10 7 / mL HepG2 2.15 cell suspension. After thorough mixing, place in a 37℃ cell culture incubator and incubate for 15 minutes. Terminate the reaction with 1×PBS, wash twice, resuspend in culture medium, count and plate. After counting NK-92 cells, add culture plates according to different effector-target ratios and incubate with CFSE-labeled HepG2 2.15 cells for 6 hours. Collect cells, wash away culture medium with 1×PBS, add 7-AAD for staining, label at room temperature for 30 minutes, and then wash for testing. The test results are as follows: Fig. 9 As shown, from Fig. 9The results showed that compared with the control group, the killing efficiency of NK92-pCDH-USP30 (OV-USP30) cells on HepG2 2.15 cells increased significantly by more than 10% at the effector-target ratio of 2.5:1 and 5:1. The supernatant of NK-92 cells was collected and co-incubated with HepG2 2.15 cells. After 24 hours, the proportion of cell apoptosis was detected by Annexin V / 7-AAD staining. The test results are shown in Figure 2. Fig.10 As shown, from Fig.10 The results of the analysis showed that the apoptotic rate of HepG2 2.15 cells treated with the supernatant of the control group was 22.19%, and the apoptotic rate of HepG2 2.15 cells treated with the supernatant of NK92-pCDH-USP30 (OV-USP30) cells was 26.69%, which was significantly higher than that of the control group.

[0093] Verification of the antiviral function of the NK-92MI cell line overexpressing USP30: The supernatant of NK-92 cells was collected and co-incubated with HepG22.15 cells. After 24 hours, the RNA of HepG22.15 cells was extracted by Trizol method, and the content of HBVcccDNA was detected by qPCR. The test results are as follows: Fig.11 As shown, from Fig.11 The analysis showed that compared with the control group, the HBV cccDNA expression level of HepG2 2.15 cells was reduced after incubation with the supernatant of NK92-pCDH-USP30 (OV-USP30).

[0094] Verification of anti-apoptosis of NK-92MI cell line overexpressing USP30 in chronic HBV infection and tumor microenvironment: The supernatant of HepG2 2.15, HepG2, and Huh7 tumor cells were collected and co-incubated with NK-92MI cells. After 24 hours, the proportion of NK-92MI cell apoptosis was detected by AnnexinV / 7-AAD staining. The test results are as follows Fig.12 , 13 As shown, from Fig.12 , 13 The results showed that the apoptotic rates of empty vector NK-92MI cells treated with supernatant of HepG22.15, HepG2 and Huh7 tumor cells were 10.7%, 16.0% and 24.4%, respectively, while the apoptotic rates of NK92-pCDH-USP30 (OV-USP30) cells treated with the same conditions were 5.63%, 3.61% and 7.54%, respectively, which were significantly lower than those of the control group.

[0095] Although the above describes the specific implementation of the present invention in combination with the embodiments, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that it should be clear to those skilled in the art that these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for improving NK cell function, characterized in that: The method is to increase the expression of deubiquitinase USP30 protein, and the method is for non-therapeutic purposes; the encoding nucleic acid of the deubiquitinase USP30 is SEQ ID NO: 1; and the product used for the expression of the deubiquitinase USP30 protein is selected from an overexpression plasmid.

2. The method for improving NK cell function according to claim 1, characterized in that: Method for expressing deubiquitinating enzyme USP30 protein NK cells were transfected by lentiviral packaging method.

3. A deubiquitinase USP30 overexpression plasmid pCDH-USP30, characterized in that: The overexpression plasmid pCDH-USP30 is a recombinant plasmid obtained by connecting the target gene USP30 as described in claim 1 to the pCDH-CMV-MCS-EF1-copGFP vector.

4. A method for constructing a NK-92 MI cell line NK92-pCDH-USP30 that overexpresses USP30 protein, characterized in that: The following steps are involved: S1. Lentivirus packaging: Transfecting the lentiviral packaging plasmids RRE, REV, VSVG and the overexpression plasmid pCDH-USP30 described in claim 3 into human embryonic kidney epithelial cells HEK293T by calcium phosphate transfection method, and performing lentiviral packaging to obtain packaged lentivirus; S2. Construction of overexpression cell lines: The packaged lentivirus obtained in S1 was concentrated, and then infected with the NK-92 MI cell line to obtain the NK-92MI cell line NK92-pCDH-USP30 overexpressing USP30.

5. The method for constructing the NK-92 MI cell line NK92-pCDH-USP30 overexpressing USP30 protein according to claim 4, characterized in that: In step S1, the amounts of lentiviral packaging plasmids RRE, REV, VSVG and recombinant plasmid pCDH-USP30 were 4.1 μg, 2.1 μg, 3.1 μg and 4.1 μg, respectively.

6. A NK-92 MI cell line NK92-pCDH-USP30 overexpressing USP30 protein, characterized in that: The method is constructed by any one of claims 4-5.

7. The use of the NK-92 MI cell line NK92-pCDH-USP30 overexpressing USP30 protein according to claim 6, characterized in that: The application is to increase the mitochondrial mass and mitochondrial membrane potential of NK92-MI cells, increase the secretion of cytokines IFN-γ and TNF-α of NK92-MI cells, increase the killing efficiency of HepG2 cells stably carrying HBV virus, namely HepG2.2.15 cells, and use it in the preparation of drugs for anti-HBV virus effects or in the preparation of drugs for reversing the exhausted state of NK cells in chronic viral infection and tumor microenvironment, wherein the chronic viral infection is chronic HBV virus infection and the tumor is liver cancer.

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