Construction and verification method of HEK293T cell line for stable overexpression of human ANT2

Abstract

The invention discloses a construction and verification method of an HEK293T cell line capable of stably overexpressing human ANT2, which comprises the following steps: cloning a human ANT2 gene, constructing the human ANT2 gene into a lentivirus expression plasmid pLVX-TRE3G, co-transfecting the lentivirus expression plasmid pLVX-TRE3G and a helper plasmid pLVX-Tet3G into an HEK293T-ACE2 cell, and carrying out puromycin and G418 pressurized screening and Western blot identification to obtain the HEK293T cell line capable of stably overexpressing human ANT2. A result shows that the expression quantity of the ANT2 is the highest when the HEK293T-ACE2-ANT2 cell is induced for 12 hours at the Doxycycline of 6mg / L; when the ANT2 is induced to express, the virus nucleic acid load and the N protein expression level after SARS-CoV-2 infection are obviously reduced, which indicates that the ANT2 has an inhibition effect on SARS-CoV-2 infection replication, and a cell model and an experimental basis are provided for exploring the effect of the ANT2 in virus infection resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of constructing HEK293T cell lines stably overexpressing human ANT2, in particular to a method for constructing and verifying HEK293T cell lines stably overexpressing human ANT2. BACKGROUND

[0002] SARS-CoV-2 invades cells by binding to the host cell surface receptor angiotensin-converting enzyme 2 (ACE2) through the spike protein (S), and ACE2 is widely distributed in the respiratory tract, cardiovascular system, kidney, liver and nervous system, thereby causing the virus to cause multiple organ damage, however, the infection and replication mechanism of SARS-CoV-2 is still not completely clear.

[0003] Previous studies have found that SARS-CoV-2 can induce an increase in the overall level of succinylation of host cells, suggesting that succinylation modification is an important means for SARS-CoV-2 to hijack host proteins. Based on the succinylation modification omics data, GO gene function annotation and functional enrichment analysis of the succinylation differential proteins induced by SARS-CoV-2 were performed, and combined with the difference in succinylation change, it was found that ANT1, ANT2 and ANT3 of the Adenine nucleotide translocase (ANT) family located in mitochondria changed significantly after SARS-CoV-2 infection, among which ANT2 was the protein with the most significant increase in succinylation level, suggesting that ANT2 has a potential regulatory role in the process of SARS-CoV-2 infection.

[0004] ANT is a class of proteins responsible for the transmembrane transport of ATP and ADP on the inner membrane of mitochondria, which widely exists in mammalian cells, insect cells and yeast cells, and this class of protein family accounts for 10% of the total amount of mitochondrial inner membrane proteins. ANT has 4 isoforms in the human body, namely ANT1, ANT2, ANT3 and ANT4, and the coding genes of the 4 isoforms are SLC25A4, SLC25A5, SLC25A6 and SLC25A31. ANT maintains the normal oxidative phosphorylation (OXPHOS) of mitochondria, homeostasis and energy supply to cells by mediating the cyclic transport of ADP and ATP between mitochondria and cytoplasm.

[0005] Some current studies show that ANT not only participates in the mitochondrial energy metabolism pathway, but also participates in the mitochondrial-related life activities, such as the processes of apoptosis, necrosis and the like, and is closely related to diseases such as cancer, infection and the like. The loss of function or overexpression of ANT can cause the imbalance of mitochondrial membrane potential and cell apoptosis, and is closely related to the pathogenic effect of viral infection.

[0006] To clarify the role of ANT2 in the process of SARS-CoV-2 infection. The present application uses a Tet-On inducible expression system to construct a HEK293T-ACE2 cell line stably overexpressing ANT2, and to verify the phenotype by infecting the cells with SARS-CoV-2, thereby providing a necessary cell model for the study of the role of ANT2 in regulating SARS-CoV-2 infection. SUMMARY

[0007] Therefore, the present application provides a method for constructing and verifying a HEK293T cell line stably overexpressing human ANT2.

[0008] To solve the above technical problems, the present application adopts the following technical solutions:

[0009] A method for constructing and verifying a HEK293T cell line stably overexpressing human ANT2, comprising the following steps:

[0010] Step 1: Construction of recombinant expression plasmid pLVX-TRE3G-ANT2

[0011] The pLVX-TRE3G plasmid is double-digested with restriction enzymes BamH I and EcoR I, and the enzyme digestion products are recovered and purified after agarose gel electrophoresis; the ANT2-Flag gene fragment is cloned from the pcDNA3.1-ANT2-Flag plasmid by high-fidelity PCR, and the reaction products are recovered and purified after agarose gel electrophoresis; the ANT2-Flag gene fragment and the double-digested pLVX-TRE3G vector fragment are mixed, Exnase MultiS and 5 × CE MultiS Buffer are added for seamless cloning connection, and then placed on ice for cooling; the competent cells are thawed on ice, the ligation products are transformed into the competent cells by heat shock method, and then plated on LB solid medium containing ampicillin for culture; the single colony with resistance is picked up in LB broth, and the plasmid is extracted after shaking, and sequencing verification is performed, and the ANT2 gene in the recombinant plasmid is identified by PCR, and the correct plasmid is named as pLVX-TRE3G-ANT2, and then the recombinant plasmid is extracted in large quantities;

[0012] Step 2: Sensitivity analysis of HEK293T-ACE2 cells to puromycin and G418

[0013] HEK293T-ACE2 cells were inoculated into cell culture plates and placed in a constant temperature incubator. After the cells adhered, 0.5, 1, 2, 3 mg / L puromycin, 1, 2, 3, 4 mg / L G418 were added according to the plan, and duplicate wells were set up;

[0014] Step 3: Construction of stable overexpression ANT2 cell line by double plasmid co-transfection

[0015] HEK293T-ACE2 cells were inoculated into cell culture plates and cultured overnight. When the cell density reached the required level, the cells were transfected. The transfected cells were subjected to pressure screening with complete culture medium containing puromycin and G418 and subcultured until no new cell death occurred, i.e., the stable cell line HEK293T-ACE2-ANT2 was obtained;

[0016] Step 4: Western blot identification of stable overexpression ANT2 cell line

[0017] HEK293T-ACE2-ANT2 cells were induced to culture in complete culture medium containing Doxycycline, and an equal amount of DMSO was used to induce the culture of cells as a control. The cells were collected to extract proteins, and the protein expression level of ANT2 was detected by Western blot;

[0018] Step 5: Optimization of expression conditions of stable overexpression ANT2 cell line

[0019] The overexpression cells were further subjected to induction expression condition optimization;

[0020] Step 6: Phenotype of stable overexpression ANT2 cell infected with SARS-CoV-2

[0021] HEK293T-ACE2-ANT2 cells were induced according to the best induction expression conditions found, and DMSO was used as a control. The viral nucleic acid and protein expression levels in the cell supernatant and cells were detected;

[0022] Step 7: Data processing and statistical analysis

[0023] The obtained experimental data were subjected to statistical analysis.

[0024] Preferably, in step 1, the seamless cloning ligation is performed at 37 °C for 30 min.

[0025] Preferably, in step 1, the competent cells are thawed on ice, 10 μL of the ligation product is transformed into 100 μL of the competent cells by heat shock method, and then plated on LB solid medium containing ampicillin and cultured at 37 °C for 12 h.

[0026] Preferably, in step 1, the plasmid is extracted after 12 h of shaking.

[0027] Preferably, in step 2, the HEK293T-ACE2 cells are inoculated into a 12-well cell culture plate.

[0028] Preferably, in step 3, the cells are transfected when the cell density reaches 80%, and at the time of transfection, the cell culture medium is first replaced with serum-free medium Opti-MEM, and then the recombinant plasmid pLVX-TRE3G-ANT2 and pLVX-Tet3G are co-transfected into HEK293T-ACE2 cells at a ratio of 1:2.

[0029] Preferably, in step 3, the transfected cells are subjected to pressure screening with complete medium containing 1 mg / L puromycin and 2 g / L G418 and subcultured.

[0030] Preferably, in step 4, the HEK293T-ACE2-ANT2 cells are induced to culture in complete medium containing 2 mg / L Doxycycline for 24 h.

[0031] Preferably, in step 5, after induction with 1, 2, 4, 6, 8, and 10 mg / L Doxycycline for 24 h, respectively, the expression levels of ANT2 under different induction doses are detected by Western blot; in addition, after induction with 2 mg / L Doxycycline for 6, 12, 24, 36, and 48 h, respectively, the expression levels of ANT2 protein after different induction times are detected by Western blot.

[0032] Preferably, in step 6, SARS-CoV-2 is infected at 0.01 MOI for 12, 24, and 48 h, respectively; in addition, SARS-CoV-2 is further infected at 0.001, 0.01, and 0.1 MOI for 48 h, respectively, and the viral nucleic acid and protein expression levels in the cell supernatant and cells are detected.

[0033] The present application has the following technical effects relative to the prior art:

[0034] (1) The present application clones the human ANT2 gene and constructs it into the lentiviral expression plasmid pLVX-TRE3G, co-transfects it into HEK293T-ACE2 cells with the helper plasmid pLVX-Tet3G, and successfully constructs a stable overexpression ANT2 cell line HEK293T-ACE2-ANT2 through puromycin and G418 pressure screening and Western blot identification;

[0035] (2) The research results of the present application show that the HEK293T-ACE2-ANT2 cell has the highest ANT2 expression amount when induced for 12 hours at 6 mg / L of Doxycycline; when ANT2 is induced and expressed, the viral nucleic acid load and N protein expression level after SARS-CoV-2 infection are significantly reduced, indicating that ANT2 has an inhibitory effect on the replication of SARS-CoV-2 infection;

[0036] (3) The present application provides a cell model and experimental basis for exploring the role of ANT2 in antiviral infection. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a construction diagram of the recombinant expression plasmid pLVX-TRE3G-ANT2 of the present application;

[0038] Among them, A is a pLVX-TRE3G plasmid double enzyme electrophoresis map, M represents Marker, 1 and 2 are both fragments after double enzyme digestion of pLVX-TRE3G;

[0039] B is an ANT2-Flag cloned gene fragment, M represents Marker, 1 and 2 are both ANT2 gene fragments;

[0040] C is a double enzyme digestion gel electrophoresis ANT2-Flag, pLVX-TRE3G fragment, M represents Marker, 1 is a blank pLVX-TRE3G plasmid control, 2 and 3 are positive clones, and 4 is a negative clone;

[0041] D is a pLVX-TRE3G-ANT2 vector schematic diagram;

[0042] Figure 2 It is a Western blot identification of stably transfected overexpression ANT2 cell line diagram of the present application;

[0043] Among them, A is a Flag tag antibody detection result diagram;

[0044] B is an ANT2 specific antibody detection result diagram;

[0045] Figure 3 It is a Western blot detection of HEK293T-ACE2-ANT2 in different mass concentrations and time ANT2 expression situation diagram of the present application;

[0046] Among them, A is the ANT2 expression amount of HEK293T-ACE2-ANT2 induced for 24 hours at different mass concentrations of Doxycycline;

[0047] B is the expression level of ANT2 of HEK293T-ACE2-ANT2 cells induced by 2 mg / L Doxycycline for different times;

[0048] Figure 4 Figure of phenotype of the stable overexpression ANT2 cell line of the application infected by SARS-CoV-2;

[0049] A is the viral nucleic acid load of HEK293T-ACE2-ANT2 cells inoculated with SARS-CoV-2 for different times with or without Doxycycline induction;

[0050] B is the viral nucleic acid load of cell supernatant of HEK293T-ACE2-ANT2 cells inoculated with SARS-CoV-2 at different MOI for 48 h with or without Doxycycline induction;

[0051] C is the intracellular viral N protein expression level of HEK293T-ACE2-ANT2 cells infected by SARS-CoV-2 for different times or infected by SARS-CoV-2 at different MOI with or without Doxycycline induction.

[0052] * indicates P<0.05, ** indicates P<0.01, and ns indicates P>0.05 (no statistically significant difference). DETAILED DESCRIPTION

[0053] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only some of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work are within the protection scope of the application.

[0054] The application discloses a method for constructing and verifying a HEK293T cell line stably overexpressing human ANT2.

[0055] 1. Experimental materials

[0056] The transgenic human embryonic kidney cell HEK293T-ACE2 expressing ACE2 is provided by Zhijidangkang (Beijing) Gene Technology Co., Ltd.

[0057] Human ANT2 gene (GenBank No: NM_001152.5) was codon-optimized and synthesized into plasmid pcDNA3.1-ANT2-Flag by Nanjing Kingsway Company. The vectors pLVX-Tet3G and pLVX-TRE3G used for the construction of stable cell lines were preserved by the Laboratory of Molecular Virology and Immunology, Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences;

[0058] Seamless cloning reagents and one-step probe qRT-PCR reagents were purchased from Novozyme Company.

[0059] Transfection reagent PEIpro was purchased from Polyplus Company.

[0060] Anti-ANT2 rabbit monoclonal antibody was purchased from Cell Signaling Technology Company.

[0061] Anti-SARS-CoV-2 N protein mouse monoclonal antibody was purchased from Yikuo Godz Company.

[0062] All operations related to SARS-CoV-2 were carried out in a biosafety laboratory.

[0063] 2. Primer design and synthesis

[0064] The primers used in the experiment were synthesized by Shanghai Sangon Biological Engineering Technology Service Co., Ltd. and are shown in Table 1.

[0065] Table 1 Primers used in the experiment

[0066] .

[0067] 3. Methods

[0068] Step 1: Construction of recombinant expression plasmid pLVX-TRE3G-ANT2

[0069] The pLVX-TRE3G plasmid was digested with restriction enzymes BamH I and EcoR I, and the digestion product was recovered and purified after agarose gel electrophoresis; the ANT2-Flag gene fragment was cloned from the pcDNA3.1-ANT2-Flag plasmid by high-fidelity PCR, and the reaction product was recovered and purified after agarose gel electrophoresis; the ANT2-Flag gene fragment and the double-digested pLVX-TRE3G vector fragment were mixed, Exnase MultiS and 5 × CE MultiS Buffer were added, and the mixture was reacted at 37 °C for 30 min for seamless cloning connection, and then cooled on ice; the competent cells were thawed on ice, 10 μL of the connection product was transformed into 100 μL of the competent cells by heat shock method, and then coated on LB solid medium containing ampicillin and cultured at 37 °C for 12 h; the resistant single colony was picked up in LB broth, and the plasmid was extracted after shaking for 12 h, and the plasmid was sent to Shanghai Biosciences Co., Ltd. for sequencing verification, and the ANT2 gene in the recombinant plasmid was identified by PCR; the correct plasmid was named pLVX-TRE3G-ANT2, and then the recombinant plasmid was extracted in large quantities.

[0070] Step 2: Analysis of the sensitivity of HEK293T-ACE2 cells to puromycin and G418

[0071] HEK293T-ACE2 cells were inoculated into a 12-well cell culture plate and placed in a constant temperature incubator. After the cells adhered, 0.5, 1, 2, and 3 mg / L of puromycin and 1, 2, 3, and 4 mg / L of G418 were added according to the plan, and a duplicate well was set. The effects of different concentrations of puromycin / G418 on the cell state were observed and recorded under a microscope every day to determine the optimal concentration.

[0072] Step 3: Construction of stable overexpression ANT2 cell line by double plasmid co-transfection

[0073] HEK293T-ACE2 cells were inoculated in a cell culture plate and cultured overnight. When the cell density reached about 80%, the cells were transfected. During transfection, the cell culture medium was replaced with serum-free medium Opti-MEM. Then the recombinant plasmid pLVX-TRE3G-ANT2 and pLVX-Tet3G were co-transfected into HEK293T-ACE2 cells at a ratio of 1:2. The transfected cells were subjected to pressure screening with complete medium containing puromycin (1 mg / L) and G418 (2 g / L) and subcultured until no new cell death occurred, and the stable cell line HEK293T-ACE2-ANT2 was obtained.

[0074] Step 4: Western blot identification of stable overexpression ANT2 cell line

[0075] HEK293T-ACE2-ANT2 cells were induced in complete medium containing 2 mg / L Doxycycline for 24 h, and an equal amount of DMSO was used to induce the control cells. The cells were collected to extract proteins, and the protein expression level of ANT2 was detected by Western blot.

[0076] Step 5: Expression condition optimization of stably overexpressing ANT2 cell lines

[0077] Further induction expression condition optimization was carried out on the identified overexpressing cells. After induction with 1, 2, 4, 6, 8, and 10 mg / L Doxycycline for 24 h, the ANT2 expression levels under different induction doses were detected by Western blot. In addition, 2 mg / L Doxycycline was used to induce for 6, 12, 24, 36, and 48 h, and the ANT2 protein expression levels after different induction times were detected by Western blot.

[0078] Step 6: Phenotype of stably overexpressing ANT2 cells infected with SARS-CoV-2

[0079] According to the best induction expression condition found, HEK293T-ACE2-ANT2 cells were induced and DMSO was used as a control. SARS-CoV-2 was infected at 0.01 MOI for 12, 24, and 48 h. In addition, SARS-CoV-2 was infected at 0.001, 0.01, and 0.1 MOI for 48 h, and the viral nucleic acid and protein expression levels in the cell supernatant and cells were detected.

[0080] Step 7: Data processing and statistical analysis

[0081] The obtained experimental data were statistically analyzed using GraphPad Prism 8.0 software. One-way ANOVA was used for multiple comparisons, and Tukey's multiple comparisons test was selected for multiple comparisons. T-test was used for comparison between two groups.

[0082] Both test methods showed significant differences at P<0.05, and extremely significant differences at P<0.01.

[0083] Example 1: Construction and identification of recombinant expression plasmid pLVX-TRE3G-ANT2

[0084] The pLVX-TRE3G plasmid was double-digested with restriction enzymes BamH I and EcoR I, and the purified enzyme digestion products were recovered by agarose gel electrophoresis.Figure 1 A) ANT2-Flag gene fragment cloned from pcDNA3.1-ANT2-Flag (B) ligation, transformation to competent cells. After broth amplification, the plasmid was extracted, identified by PCR amplification and agarose gel electrophoresis, and the results showed that there was a band at 989 bp, which was consistent with the expected size (C), and no other bands, indicating that the ANT2 eukaryotic expression plasmid pLVX-TRE3G-ANT2 was successfully constructed (D). Figure 1 Figure 1 Figure 1

[0085] Example 2: Sensitivity analysis of HEK293T-ACE2 cells to puromycin and G418

[0086] Before cell screening, the optimal mass concentration of the two screening antibiotics needs to be determined.

[0087] The cells were treated with different mass concentrations of puromycin and G418, respectively, and their survival conditions were observed and recorded.

[0088] When the mass concentration of puromycin was 1 mg / L and that of G418 was 2 g / L, almost all the cells floated within 72 hours.

[0089] Therefore, the concentrations of puromycin and G418 were set to 1 mg / L and 2 g / L, respectively.

[0090] Example 3: Identification of stable overexpression ANT2 cell line

[0091] After co-transfecting the recombinant plasmid with pLVX-Tet3G into cells and screening with antibiotics, the stable cell line HEK293T-ACE2-ANT2 was obtained. The cell pellet was collected, total protein was extracted, and ANT2 expression was detected. The results of the induced expression verification showed that Doxycycline could effectively induce HEK293T-ACE2-ANT2 cells to overexpress ANT2. Both Flag-tagged antibody and ANT2-specific antibody could detect it, where A. is the detection result of Flag-tagged antibody, and B. is the detection result of ANT2-specific antibody; indicating that the HEK293T-ACE2 cell line overexpressing ANT2 induced by Doxycycline has been established.

[0092] Example 4: Optimization of expression conditions of stable overexpression ANT2 cell line

[0093] The correctly identified stable cell line HEK293T-ACE2-ANT2 was optimized for expression from the induction mass concentration and time.

[0094] ​​​ANT2-Flag expression was detected by Western Blot at 6, 12, 24, 36, and 48 h after induction with different concentrations of Doxycycline.

[0095] The results are shown in Figure 3 A and Figure 3 A, wherein A is the expression of ANT2 in HEK293T-ACE2-ANT2 cells induced with different concentrations of Doxycycline for 24 h; and B is the expression of ANT2 in HEK293T-ACE2-ANT2 cells induced with 2 mg / L Doxycycline for different times.

[0096] It can be seen that the highest expression of ANT2 was obtained under the induction condition of 6 mg / L Doxycycline. The expression of ANT2 began to increase significantly at 12 h, and reached the highest at 12 h. Therefore, the highest induction expression condition of ANT2 was 6 mg / L Doxycycline for 12 h.

[0097] Example 5: Phenotype of SARS-CoV-2 infection in stable overexpression of ANT2 cells

[0098] To confirm the effect of endogenous ANT2 expression on SARS-CoV-2 infection, HEK293T-ACE2-ANT2 cells were induced with 6 mg / L Doxycycline or DMSO (as a control) for 12 h under the highest induction condition, and then infected with SARS-CoV-2 at different MOIs and times.

[0099] The results showed that the viral load in the supernatant of HEK293T-ACE2-ANT2 cells infected with SARS-CoV-2 for 24 h was significantly reduced (P<0.05) (A) compared with the DMSO control group after Doxycycline induction. Moreover, the viral load was significantly reduced (P<0.05 or P<0.01) (B) in SARS-CoV-2 infection at different MOIs. The expression of viral N protein in infected cells showed a consistent trend with the nucleic acid level, and the expression of viral N protein was significantly reduced in Doxycycline-induced cells after 24 h of infection (C). Figure 4 Figure 4 Figure 4

[0100] ​​​In summary, the Tet-On system used for constructing the stable cell line of the application is a tetracycline-induced conditional gene expression system, which is composed of a regulatory expression vector pLV-Tet3G and a responsive expression vector pLV-TRE3G double plasmid; the expression system has the advantages of safety, high efficiency, controllability and the like.

[0101] The HEK293T-ACE2-ANT2 stable overexpression cell constructed by the application can overexpress ANT2 under the induction of Doxycycline, and the overexpression conditions of HEK293T-ACE2-ANT2 are optimized with different induction doses and times, and the optimal induction conditions of the cell overexpressing ANT2 are determined;

[0102] By endogenous expression of ANT2 protein in host cells, it is found that ANT2 has the effect of inhibiting the infection and replication of SARS-CoV-2, which establishes a foundation for further analyzing the infection pathogenic mechanism of SARS-CoV-2 hijacking the cell metabolic pathway.

[0103] The above is only a preferred embodiment of the application, and does not limit the technical scope of the application in any way, so any slight modification, equivalent change and modification made according to the technical essence of the application to the above embodiment are still within the scope of the technical solution of the application.

Claims

1. A method for constructing and verifying a HEK293T cell line stably overexpressing human ANT2, characterized in that, Comprising the following steps: Step 1: Construction of recombinant expression plasmid pLVX-TRE3G-ANT2 The pLVX-TRE3G plasmid was double-digested with restriction enzymes BamH I and EcoR I, and the digested product was recovered and purified after agarose gel electrophoresis; The ANT2-Flag gene fragment was cloned from the pcDNA3.1-ANT2-Flag plasmid by high-fidelity PCR, and the reaction product was recovered and purified after agarose gel electrophoresis; the ANT2-Flag gene fragment and the double-digested pLVX-TRE3G vector fragment were mixed, Exnase MultiS and 5 × CE MultiS Buffer were added for seamless cloning connection, and then the mixture was cooled on ice; the competent cells were thawed on ice, the connection product was transformed into the competent cells by heat shock method, and then coated on LB solid medium containing ampicillin for culture; resistant single colonies were picked on LB broth, and the plasmid was extracted after shaking, and sequencing verification was performed; the ANT2 gene in the recombinant plasmid was identified by PCR, and the correct plasmid was named pLVX-TRE3G-ANT2; then a large amount of recombinant plasmid was extracted; Step 2: Analysis of HEK293T-ACE2 cell sensitivity to puromycin and G418 HEK293T-ACE2 cells were inoculated into cell culture plates and placed in a constant temperature incubator; after the cells adhered, 0.5, 1, 2, and 3 mg / L of puromycin and 1, 2, 3, and 4 mg / L of G418 were added according to the plan, and a duplicate well was set; Step 3: Construction of stable overexpression ANT2 cell line by double plasmid co-transfection HEK293T-ACE2 cells were inoculated in cell culture plates and cultured overnight; when the cell density reached the required value, the cells were transfected; the transfected cells were subjected to pressure screening with complete medium containing puromycin and G418 and subcultured until no new cell death occurred, and the stable cell line HEK293T-ACE2-ANT2 was obtained; Step 4: Western blot identification of stable overexpression ANT2 cell line HEK293T-ACE2-ANT2 was induced to culture in complete medium containing Doxycycline, and an equal amount of DMSO was used to induce the culture of cells as a control; the cells were collected to extract proteins, and the protein expression level of ANT2 was detected by Western blot; Step 5: Optimization of expression conditions of stable overexpression ANT2 cell line The identified overexpression cells were further subjected to induction expression condition optimization; Step 6: Phenotype of stable overexpression ANT2 cell infected with SARS-CoV-2 HEK293T-ACE2-ANT2 cells were induced according to the best induction expression conditions, and DMSO was used as a control; the viral nucleic acid and protein expression levels in the cell supernatant and cells were detected; Step 7: Data processing and statistical analysis The obtained experimental data were subjected to statistical analysis.

2. The method according to claim 1, wherein the method is characterized by, In step 1, the seamless cloning connection was performed at 37 °C for 30 min.

3. The method for constructing and validating a stable HEK293T cell line overexpressing human ANT2 according to claim 1, characterized in that, In step 1, the competent cells were thawed on ice, 10 μL of the ligation product was transformed into 100 μL of the competent cells by heat shock, and then spread on LB solid medium containing ampicillin and incubated at 37 ℃ for 12 h.

4. The method according to claim 1, wherein the method is characterized by, In step 1, the plasmid was extracted after 12 h of shaking.

5. The method according to claim 1, wherein the method is characterized by, In step 2, the HEK293T-ACE2 cells were inoculated into a 12-well cell culture plate. 6.The method of claim 1, wherein the method is characterized by, In step 3, when the cell density reached 80%, the cells were transfected. At the time of transfection, the cell culture medium was first replaced with serum-free medium Opti-MEM, and then the recombinant plasmid pLVX-TRE3G-ANT2 and pLVX-Tet3G were co-transfected into HEK293T-ACE2 cells at a ratio of 1:

2.

7. The method according to claim 1, wherein the method is characterized by, In step 3, the transfected cells were subjected to pressure screening with complete medium containing 1 mg / L puromycin and 2 g / L G418 and subcultured. 8.The method of claim 1, wherein the method is characterized by, In step 4, the HEK293T-ACE2-ANT2 cells were induced in complete medium containing 2 mg / L doxycycline for 24 h. 9.The method of claim 1, wherein the method is characterized by, In step 5, after induction with 1, 2, 4, 6, 8, and 10 mg / L doxycycline for 24 h, respectively, the expression levels of ANT2 under different induction doses were detected by Western blot. In addition, after induction with 2 mg / L doxycycline for 6, 12, 24, 36, and 48 h, respectively, the expression levels of ANT2 protein after different induction times were detected by Western blot. 10.The method of claim 1, wherein the method is characterized by, In step 6, SARS-CoV-2 was infected at 0.01 MOI for 12, 24, and 48 h, respectively. In addition, SARS-CoV-2 was infected at 0.001, 0.01, and 0.1 MOI for 48 h, respectively, and the viral nucleic acid and protein expression levels in the cell supernatant and cells were detected.

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