Investigation of Interaction between the Spike Protein of SARS-CoV-2 and ACE2-Expressing Cells Using an In Vitro Cell Capturing System

Cell Culture

ALL cell lines used in this study were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HEK293T (human, kidney), Huh-7 (human, liver), MRC-5 (human, lung), U251M (human, brain) cells were cultured in DMEM (Dulbecco’s’ modified Eagle medium) (Hyclone), H1299 AND H460 (human, lung) cells were cultured in RPMI-1640(Hyclone), MRC5(human, lung) and HepG2 (human, lung) cells were cultured in MEM (Minimum Essential Medium, Gibco) at 37 °C and 5% CO2 in a humidified atmosphere, supplemented with 10% fetal bovine serum (FBS) and penicillin (100 μg/ml), streptomycin sulfate (100 μg/ ml), 1x non-essential amino acid solution (10x stock, PAA) and 10 mM sodium pyruvate (ThermoFisher Scientific). For normal seeding and subcultivation, cells were first washed with phosphate buffered saline (PBS) and then incubated in the presence of trypsin/EDTA solution (Cytiva) until the cells detached. For capturing, cells were detached with Enzyme-Free Cell Dissociation Solution (ThermoFisher).

Plasmids

Expression plasmids ofSARS-CoV-2 spike (QHD43416.1) and S1 with or without C-terminal HA epitope tag were purchased from Sino Biological. human ACE2 tagged with GFP (ACE2-GFP) was purchased from Sino Biological (HG10108-ACG).

Construction of Cell Line with Stable ACE2 Expression

HEK293T cells were transfected with pCMV-ACE2-GFPSark tag expression plasmid by lipo3000 according to manual procedure, Hygromycin B selected (110 μg/ml) for 7d and analyzed for ACE2-GFP expression by Western blot and immunostaining using an antibody to ACE2 (ACE2–10108-T24, Sino Biological).

qRT-PCR Analysis

Total RNA of 293 T, Huh7, HepG2, H1299, H460, U251 and MRC5 cells were isolated using TransZol Up plus RNA Kit (TIANGEN). cDNA synthesized through a PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa) and using random hexamers as primers (42 °C for 2 min, 37 °C for 15 min, 85 °C for 5 s). Forward primer GACAAGAGCAAACGGTTGAACAC and reverse primer GCCCAGAGCCTCTCATTGTAG were used for the qRT-PCR for ACE2. Genes were analyzed using Realstar Green Fast Mixture UNG with ROX (GenStar A305–05) on a LightCycler 480 Real-Time PCR system (Roche). Internal H1299 mRNA levels were used for normalization and the fold expression was calculated using the 2−ΔCT method compared to the 293 T, Huh7, HepG2, H1299, H460, U251, MRC5 cells mRNA levels.

Western Blotting

HEK293T, Huh7, HepG2, H1299, H460, U251 and MRC5 cells were harvested, washed with PBS and subsequently lysed in RIPA Lysis and Extraction Buffer (Thermo Fisher). Total protein levels were determined by a bicinchoninic acid assay (ThermoFisher), and 10 μg of protein was resolved by 12% SDS polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose, blocked in 5% bovine serum albumin (BSA), and incubated with anti-ACE2 antibody (10108-T24, Sino Biological) or anti-β tubulin (sc-166,729; Santa Cruz). Protein was detected using horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Amersham) and Luminata Forte Western HRP substrate (Millipore).

Co-IP

HEK293T/ACE2 stable cell line was generated by transducing HEK293T cells with pCMV-Flag-ACE2,(Sino Biological, HG10108-NF), and 1 × 106 cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate), supplemented with protease inhibitors. Lysates were first incubated with S1 or S1(D614G) for 4 h at 4 °C, then incubated with anti-FLAG antibody (M2, Sigma-Aldrich) or mouse IgG conjugated to Dynabeads Protein G (Thermo Fisher Scientific) overnight at 4 °C. The beads were washed with wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate) five times. Eluted protein samples were separated by SDS–polyacrylamide gel electrophoresis, and immunoprecipitated with anti-Flag antibody (Sigma, F7425) or anti-spike antibody (ThermoFisher, PA5–81795).

Cell Capture and Detached by Immobile S1

96-well ELISA plates (CIH-F8T, GSBIO) were coated with 0.5 μg/well of spike S1-his protein in PBS buffer (pH 7.0) overnight at 4 °C. After washing with 0.05% Tween 20-PBS (w/v), wells were and blocked with 3% BSA-PBS (w/v) for 1 h. For cell capturing, 3 104 cells were added to each S1-coated well, incubated for 15 min at 37 degree, then washed 5 times with PBS (containing 1% BSA). To quantify captured cells, three methods were utilized. The first one is the CCK8 test, where captured cells were washed once with 300 μl PBS, incubated with 100 μl CCK8 working solution (complete medium: CCK8 = 10:1) at 37 °C, 5% CO2 for 3 h. Subsequently, OD values at 450 nm were determined and number of cells was calculated according to the standard curve. Alternatively, cells were detached by using Trypzin-EDTA then counted by flow cytometry. In case number of captured cells was low, captured cells were counted manually under the Optical microscope.

Quantification of Cell Viability

Cell viability of captured cells was analyzed using the Annexin V-FITC Apoptosis Detection Kit (Beyotime). In brief, the captured cells were digested with trypsin, centrifuged at 300 g for 5 min and washed twice with PBS. The cell pellet was gently resuspended with 195 UL binding solutions, stained with Annexin V-FITC (5ul) and propidium iodide (10 UL) in the dark for 10-20 min, then immediately deterimined using flow cytometry.

Immunofluorescence Staining

ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen). After staining, slides were mounted using ProLong Antifade (Thermo Fisher) solution and observed using a Leica SP8 confocal microscope.

Mice Anti-Spike Serum Sample Preparation

Eight-week-old female BALB/c mice were obtained from Weitonglihua Co. Limited (Beijing, China) and housed in a specific pathogen-free (SPF) animal facility. Mice were immunized with 50 μg of SARS-Cov-2 spike protein (40607-V08B, Sino Biological) emulsified incomplete Freund’s adjuvant (CFA) by subcutaneous injection of up to 100 μl volume per site. Two and 4 weeks after the first immunization, mice were boosted another with S protein emulsified in Incomplete Freund’s adjuvant (IFA). Serum samples from immunized mice or control mice were prepared and tested by ELISA with a minimum dilution of 100-fold to confirm antibody response.

Statistical Analysis

Results shown in each figure were derived from at least three independent experiments with comparable findings. The comparison of means between two groups was conducted using Student’s t-test, whereas comparison for more than two groups was conducted using one-way ANOVA. Only p values of 0.05 or lower were considered statistically significant. All statistical analyses were performed using the GraphPad Prism 7 software package (GraphPad Software).

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