Establishment of a renal fibrosis model induced by SARS-CoV-2 envelope protein

Abstract

The present invention belongs to the field of biotechnology and relates to the establishment of a SARS-CoV-2 envelope protein-induced renal fibrosis model. Specifically, it includes the establishment of a SARS-CoV-2 envelope protein-induced mouse renal fibrosis model and the establishment of a SARS-CoV-2 envelope protein-induced HK-2 cell epithelial-mesenchymal transition model. The present invention discloses for the first time a method for establishing a SARS-CoV-2 envelope protein-induced renal fibrosis model, which solves the problems of high cost and harsh operating conditions in the existing SARS-CoV-2 direct infection model establishment method, and provides a new experimental approach for further in-depth research on SARS-CoV-2-related renal pathological mechanisms and screening of related drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the establishment of a SARS-CoV-2 envelope protein-induced renal fibrosis model, specifically the establishment of a SARS-CoV-2 envelope protein-induced mouse renal fibrosis model and the establishment of a SARS-CoV-2 envelope protein-induced HK-2 cell epithelial-mesenchymal transition model. Background Art

[0002] The new coronavirus (SARS-CoV-2) has seriously endangered human health. In addition to infecting the respiratory system, SARS-CoV-2 can also damage multiple organs throughout the body. Among them, the kidney is one of the main damaged organs. SARS-CoV-2 can directly infect renal cells. The infected kidneys mainly show tubular damage, and tubular interstitial fibrosis-related indicators were significantly increased in the patient's autopsy samples. Clinically, kidney damage in SARS-CoV-2 infected people will not only increase the difficulty of treatment and care, but also increase the mortality rate of people with underlying kidney disease. A SARS-CoV-2-related renal fibrosis model is of great significance for the in-depth study of SARS-CoV-2. However, the establishment of a SARS-CoV-2 direct infection model has the following two difficulties: (1) SARS-CoV-2 cannot infect rodents, and humanized mice are required, which greatly increases the cost of the model; (2) SARS-CoV-2 needs to be operated in a biosafety level 3 laboratory, which has strict operating requirements and cannot be achieved in most laboratories. Therefore, it is of great significance to establish an animal model of renal fibrosis similar to SARS-CoV-2 direct infection. The fibrosis of animal organs corresponds to epithelial-mesenchymal transition at the cellular level. The transformation of epithelial cells to mesenchymal cells caused by a series of pathological factors is the basis for the occurrence of fibrosis. Therefore, it is also of great significance to establish an epithelial-mesenchymal transition model similar to direct infection with SARS-CoV-2 at the cellular level. Summary of the invention

[0003] The purpose of the present invention is to provide a SARS-CoV-2 envelope protein-induced mouse renal fibrosis model and a SARS-CoV-2 envelope protein-induced HK-2 cell epithelial-mesenchymal transition model, which solves the problems of high cost and harsh operating conditions in the existing SARS-CoV-2 direct infection model establishment method.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0005] The first aspect of the present invention discloses the application of SARS-CoV-2 envelope protein in inducing the establishment of a renal fibrosis model.

[0006] Among them, the SARS-CoV-2 envelope protein needs to be purified and extracted through the pET28a-SARS-CoV-2-E-6×His plasmid prokaryotic expression system.

[0007] Preferably, the prokaryotic expression system is the Escherichia coli BL21 DE3 pLysS expression system.

[0008] Specifically, SARS-CoV-2 protein expression was induced with 0.5 mM isopropyl-bD-1-thiogalactopyranoside at 22°C for 16-18 hours, and then SARS-CoV-2 envelope protein was purified.

[0009] Preferably, the specific method of purification is: using Ni-NTA resin to separate 6×His envelope protein and using endotoxin removal agarose resin to remove endotoxin.

[0010] Renal fibrosis models include animal models and cell models:

[0011] The animal model uses rodents, such as mice, and the purified envelope protein is injected into the mice through the tail vein (25 mg kg -1 ), one injection, during which urine was collected to detect urine creatinine and urine protein levels, and renal function was monitored. After about 4 weeks, obvious renal fibrosis appeared, and a renal fibrosis animal model was obtained;

[0012] Preferably, wild-type mice can be used to establish the animal model in the present invention.

[0013] Preferably, the establishment of the animal model in the present invention only requires one tail vein injection.

[0014] Preferably, the establishment of the animal model in the present invention only takes 4 weeks.

[0015] The cell model used human renal tubular epithelial cells, the HK-2 cell line. After the HK-2 cells were serum-starved for 12 hours, the purified envelope protein (2 μg / mL) was added to the HK-2 cell culture medium and cultured for 24 hours. The HK-2 cells showed obvious epithelial-mesenchymal transition, and a renal fibrosis cell model was obtained.

[0016] Preferably, the establishment of the cell model in the present invention only requires adding the inducer once, without any other special operations.

[0017] In the cell model established by the present invention, epithelial-mesenchymal transition-related indicators are significantly upregulated.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The technical solution of the present invention shows that the envelope (E) protein, as one of the main structural proteins of SARS-CoV-2, can act as an independent virulence factor to cause inflammation and multi-organ damage. Moreover, the type of damage induced by the envelope protein is highly similar to that caused by direct infection with SARS-CoV-2, and can be a good substitute for studying the pathological process related to SARS-CoV-2.

[0020] The present invention further provides a method for establishing an animal and cell model of renal fibrosis by directly infecting an envelope protein, a structural protein of SARS-CoV-2. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 .Effects of envelope protein on renal function and body weight of mice:

[0022] (A) Schematic diagram of animal modeling: Mock group (n = 6), mice were injected with TBS (purified protein eluate) in the same volume as the purified protein through the tail vein; E Protein group (n = 6), mice were injected with envelope protein (25 mg kg -1 Body weight); (B) renal function assessment (n=6), proteinuria was quantified by the ratio of urine protein to creatinine; (C) changes in mouse body weight during the experiment (n=6).

[0023] Figure 2 .Effects of envelope protein on the pathological structure of mouse kidney:

[0024] (A) Representative images of kidney pathological lesions; (B) Representative images of kidney hematoxylin-eosin (H&E) staining (scale bar = 50 μm).

[0025] Figure 3 .Envelope protein induces renal fibrosis in mice:

[0026] (A) Effect of envelope protein on protein expression of renal fibrosis indicators (fibronectin (FN), vimentin, and α-smooth muscle actin (α-SMA)); (B) Quantitative analysis of protein expression levels; (C) Effect of envelope protein on mRNA levels of fibrosis indicators (fibronectin (FN), vimentin, and α-smooth muscle actin (α-SMA)); (D) Representative images of kidney Masson trichrome staining (scale bar = 50 μm). All values ​​are mean ± SD; one-way ANOVA. **P < 0.01, ***P < 0.001, (n = 6).

[0027] Figure 4 .Envelope protein induces epithelial-mesenchymal transition of HK-2 cells:

[0028] (A) Effect of envelope protein on the expression of epithelial-mesenchymal transition indicators (fibronectin (FN), vimentin, and α-smooth muscle actin (α-SMA)); (B) Quantitative analysis of protein expression levels. All values ​​are mean ± SD; one-way ANOVA. ns P>0.05, *P<0.05, ***P<0.001, (n=5).

[0029] Figure 5 .Envelope protein activates the TGF-β1 / Smad2 / 3 pathway by stimulating the secretion of TGF-β1 in mouse kidneys:

[0030] (A) Levels of TGF-β1 in mouse serum. (B) Effects of envelope protein on activation of TGF-β1 / Smad2 / 3 pathway in kidney. (C) Quantitative analysis of protein expression levels. All values ​​are mean ± SD; one-way ANOVA. ***P < 0.001, (n = 6).

[0031] Figure 6 .Envelope protein activates TGF-β1 / Smad2 / 3 pathway by stimulating the secretion of TGF-β1 in HK-2 cells:

[0032] (A) Levels of TGF-β1 in the supernatant of HK-2 cells. (B) Effects of envelope proteins on the activation of TGF-β1 / Smad2 / 3 pathway in HK-2 cells. (C) Quantitative analysis of protein expression levels. All values ​​are mean ± SD; one-way ANOVA. ***P < 0.001, (n = 5). DETAILED DESCRIPTION

[0033] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications. The reagents and raw materials used in the present invention are all commercially available.

[0034] Example 1

[0035] This example discloses a method for establishing a mouse renal fibrosis model induced by SARS-CoV-2 envelope protein, as follows:

[0036] 1. Animals

[0037] C57BL / 6 mice, male, 8 weeks old, were purchased from Shanghai Lingchang Experimental Animal Co., Ltd. and housed in the SPF-grade animal laboratory of the Experimental Animal Center of the School of Pharmacy, Fudan University.

[0038] 2. Experimental Materials

[0039] Anti-α-SMA (1:10000) and FN (1:1000) antibodies were purchased from Abcam. Anti-GAPDH (1:5000), p-Smad23 (1:10001), Smad23 and vimentin (1:1000) antibodies were purchased from Cell Signaling Technology (CST). Anti-TGF-β1 (1:1000) was purchased from Ibotek.

[0040] 3. Experimental instruments

[0041] Biological safety cabinet (Thermo Fisher), CO2 cell culture incubator (Thermo Fisher), refrigerated centrifuge (Thermo Fisher), micropipette (Eppendorf), constant temperature water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.), gel electrophoresis apparatus (Bio Rad), transfer apparatus (Bio Rad), gel image processing system (BioRad), inverted microscope (Carl Zeiss), Q-RTPCR instrument (Bio-Rad), tissue homogenizer (Shanghai Jingxin Technology), and microplate reader (Tecan Systems Inc.).

[0042] 4. Experimental methods

[0043] (1) Protein purification

[0044] Induced expression: pET28a-SARS-CoV-2-E-6×His plasmid was transfected into E. coli BL21(DE3)pLysS (TransGen Biotech) to express the fusion protein. -1 A single colony was started in 50 mL LB medium containing kanamycin and the cells were cultured at 37°C with shaking at 220 rpm. The initial culture was inoculated into 1 L LB medium (containing 50 μg mL -1 Kanamycin), 37°C, 220 rpm shaking culture, until the optical density at 600 nm (OD600) reaches 0.6-0.8. Then add 0.5 mM IPTG (isopropyl-bD-1-thiogalactopyranoside; Yeasen), 22°C, 220 rpm, and induce protein expression for 16-18 hours.

[0045] Preparation of protein suspension: 4 ° C, 8000rpm centrifugation for 20 minutes to collect bacteria, and resuspend them in binding buffer (150mMNaCl, 20mM Tris-base, 1mM DTT, 1%PMSF, 1×cocktail, pH 8.0). The resuspended bacteria were lysed by sonication at 4 ° C for 60 minutes. The soluble part of the cell lysate was separated from the cell debris by centrifugation at 10000g for 30 minutes at 4 ° C, and the supernatant was filtered through a 0.22μM filter to remove particles.

[0046] Separation and purification: Ni-NTA resin (Yeasen) was pre-equilibrated with TBS buffer (150mM NaCl, 20mM Tris-base, pH 8.0). The clarified supernatant was loaded on 5mL Ni-NTA for 4 hours to separate E-6×His fusion protein from E. coli protein. The column was washed with 10 column volumes with TBS eluents containing 20mM, 30mM, and 50mM imidazole concentrations, respectively. After washing, the E-6×His fusion protein was eluted with TBS eluent containing 300mM imidazole. The E-6×His fusion protein was then purified using Superdex 75Increase 10 / 300 gel filtration chromatography (GE Healthcare) pre-equilibrated with TBS. Finally, endotoxin was removed with endotoxin removal agarose resin (Yeasen).

[0047] Concentration: After equilibration of the agarose resin with TBS, the purified protein was concentrated at 0.25 mL min -1 The eluate was collected and concentrated using a centrifugal filter device (Amicon@Ultra).

[0048] (2) Establishment of a mouse renal fibrosis model induced by SARS-CoV-2 envelope protein

[0049] Twelve 8-week-old male C57BL / 6 mice were randomly divided into 2 groups: control group (n=6), which was injected with TBS (purified protein eluate) in the same volume as the purified protein through the tail vein; envelope protein group (n=6), which was injected with purified envelope protein (25 mg kg -1 weight)

[0050] (3) Renal function assessment

[0051] Urine samples were collected during the modeling period, and renal function was evaluated by the ratio of urine protein to creatinine. Urine protein content was measured by a urine protein detection kit (Nanjing Jiancheng Bioengineering Institute, China). Creatinine content was measured using a creatinine determination kit (Nanjing Jiancheng Bioengineering Institute, China).

[0052] (4) Kidney pathology detection

[0053] Mouse kidney tissues were isolated and fixed in 10% formalin at room temperature for 48 hours. The fixed tissues were embedded in paraffin and cut into 4 μm thick sections for hematoxylin-eosin (H&E) and Masson's trichrome staining.

[0054] (5) Detection of cytokine TGF-β1

[0055] Mouse serum was centrifuged at 1500 g for 10 min at 4°C, and the supernatant was collected. Cytokines in mouse serum were measured using an ELISA kit (Multi Science Biotech) according to the instructions.

[0056] (6) Protein expression detection

[0057] Proteins in kidney were analyzed by Western blotting. Kidney tissue was lysed on ice for 30 minutes with RIPA buffer supplemented with 1% mixed protease inhibitors (Beyotime). Insoluble materials were removed by centrifugation at 12000 g for 15 minutes, and the supernatant was collected.

[0058] Protein was quantified using BCA protein assay (Beyotime) using BSA as standard. SDS-PAGE sample loading buffer (Beyotime) was added to the sample protein and denatured at 95°C for 5-10 minutes. Proteins of various molecular weights were separated by SDS-PAGE, transferred to PVDF membranes (Millipore), blocked with 5% fat-free milk in TBST buffer (0.1% Tween-20 and 0.1M NaCl, 0.1M Tris-HCl, pH 7.5) for 1 hour at room temperature, and then incubated with primary antibodies overnight at 4°C. After washing three times with TBST (10 minutes each time), horseradish peroxidase-conjugated secondary antibodies (1:5000; SantaCruz Biotechnology) were added and incubated at room temperature for 1 hour. Protein bands were quantitatively analyzed by using an image analysis system (Quantity One software; BioRad ChemidDoc, BioRad, USA).

[0059] (7) mRNA detection

[0060] Total RNA was extracted from kidney using TRIzol reagent (Invitrogen), and complementary DNA was synthesized using primeScript RT Master Mix (Takara). qRT PCR amplification was performed using TB Green premix Ex Taq (Takara) according to the instructions. The results were normalized using the GAPDH gene as an internal reference, and the fold change was calculated using the 2-(ΔΔCt) formula.

[0061] The experimental results are as follows Figure 1 , 2 As shown in Figures 3 and 5, envelope protein can damage renal tubular cells, causing weight loss, proteinuria, and obvious renal fibrosis. The mechanism of inducing renal fibrosis may be through promoting the secretion of TGF-β1, thereby activating the TGF-β1 / Smad2 / 3 pathway.

[0062] Example 2

[0063] This example discloses a method for establishing an epithelial-mesenchymal transition model of HK-2 cells induced by SARS-CoV-2 envelope protein.

[0064] The experimental method is as follows:

[0065] (1) Establishment of the epithelial-mesenchymal transition model of HK-2 cells induced by SARS-CoV-2 envelope protein

[0066] HK-2 (human renal proximal tubule epithelial cells) were cultured in RPMI1640 medium (Thermo Fisher Scientific) containing 10% serum (Gibco) and 100 U·mL-1 penicillin / streptomycin (Gibco).

[0067] 5×105HK-2 cells were seeded in 6-well plates with complete medium for 12 hours. Then, they were starved in serum-free medium for 12-16 hours. After starvation, the cells were stimulated with purified envelope protein at a series of concentrations (0.5, 1, 2 μg / mL) for 24 hours.

[0068] (2) Detection of cytokine TGF-β1

[0069] The cell culture supernatant was centrifuged at 1500 g for 10 min at 4°C and the supernatant was collected. Cytokines were measured using an ELISA kit (Multi Science Biotech) according to the manufacturer's instructions.

[0070] (6) Protein expression detection

[0071] Proteins in cells were analyzed by Western blotting. Kidney tissue was lysed on ice for 30 minutes with RIPA buffer supplemented with 1% mixed protease inhibitors (Beyotime). Insoluble materials were removed by centrifugation at 12000 g for 15 minutes, and the supernatant was collected.

[0072] Protein was quantified using BCA protein assay (Beyotime) using BSA as standard. SDS-PAGE sample loading buffer (Beyotime) was added to the sample protein and denatured at 95°C for 5-10 minutes. Proteins of various molecular weights were separated by SDS-PAGE, transferred to PVDF membranes (Millipore), blocked with 5% fat-free milk in TBST buffer (0.1% Tween-20 and 0.1M NaCl, 0.1M Tris-HCl, pH 7.5) for 1 hour at room temperature, and then incubated with primary antibodies overnight at 4°C. After washing three times with TBST (10 minutes each time), horseradish peroxidase-conjugated secondary antibodies (1:5000; SantaCruz Biotechnology) were added and incubated at room temperature for 1 hour. Protein bands were quantitatively analyzed by using an image analysis system (Quantity One software; BioRad ChemidDoc, BioRad, USA).

[0073] (7) mRNA detection

[0074] Total RNA was extracted from cells using TRIzol reagent (Invitrogen), and complementary DNA was synthesized using primeScript RT Master Mix (Takara). qRT PCR amplification was performed using TB Green premix Ex Taq (Takara) according to the instructions. The results were normalized using the GAPDH gene as an internal reference, and the fold change was calculated using the 2-(ΔΔCt) formula.

[0075] The experimental results are as follows Figure 4 , 6 As shown: Envelope protein can induce HK-2 cells to undergo obvious epithelial-mesenchymal transition, and the related mechanism of inducing epithelial-mesenchymal transition may be through promoting the secretion of TGF-β1, thereby activating the TGF-β1 / Smad2 / 3 pathway. The related mechanism is similar to that of animal models.

[0076] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for establishing an animal model of renal fibrosis induced by SARS-CoV-2 envelope protein, characterized in that: The specific method is as follows: The purified envelope protein was injected into wild mice via the tail vein and a renal fibrosis animal model was obtained 4 weeks later. The dosage of purified protein was 25 mg∙kg -1 ; The SARS-CoV-2 envelope protein was purified and extracted by the pET28a-SARS-CoV-2-E-6×His plasmid prokaryotic expression system.

2. The method according to claim 1, characterized in that The prokaryotic expression system is Escherichia coli E. coli BL21DE3 pLysS expression system.

3. The method according to claim 2, characterized in that SARS-CoV-2 protein expression was induced with 0.5 mM isopropyl-bD-1-thiogalactopyranoside at 22°C for 16-18 hours, and then SARS-CoV-2 envelope protein was purified.

4. The method according to claim 3, characterized in that The specific method of the purification is: using Ni-NTA resin to separate 6×His envelope protein and using endotoxin removal agarose resin to remove endotoxin.

5. A method for establishing a renal fibrosis cell model using SARS-CoV-2 envelope protein induction, characterized in that: The specific method is as follows: Human renal tubular epithelial cells, HK-2 cell line, were used. After HK-2 cells were serum-free for 12 hours, the purified envelope protein was added to the HK-2 cell culture medium and cultured for another 24 hours. HK-2 cells showed obvious epithelial-mesenchymal transition, and a renal fibrosis cell model was obtained. The concentration of the envelope protein in the culture medium is 0.5-2 μg / mL.

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