Our research is focused on understand of biology of SARS-CoV-2 virus and try to find drugs, particularly from Chinese medicine against SARS-CoV-2. All the live SARS-CoV-2 virus study must be controlled at a biosafety level three lab. And this experimental limitation make a SARS-CoV-2 study only feasible in a handful of lives. The SARS-CoV-2 virus like practical method of the study about SARS-CoV-2 possible without limitation of biosafety level three lab, and the list will be a very useful method.
[Instructor] To begin, seed approximately 3 million HEK-293T cells in a 10-centimeter diameter tissue culture plate with DMEM complete medium, supplemented with 10% FBS and 1% penicillin-streptomycin. Culture the cells at 37 degrees Celsius and 5% carbon dioxide for approximately 24 hours. Check the cell co fluency under the microscope. Dilute 60 microliters of PEI from a one milligram per milliliter stock solution with serum-free medium to reach a final volume of 200 microliters. Now, take 200 microliters of serum-free medium and add 6.7 micrograms of N plasmid, 10 micrograms of Luc-T20 plasmid, 0.016 micrograms of S plasmid, and 3.3 micrograms of M-IRES-E plasmid into it. Gently add the diluted PEI solution into the solution containing plasmids coating for viral structure proteins and incubate the mixture at room temperature for 10 minutes. This is the transfection solution. Carefully drop the transfection solution onto the HEK-293T cells and gently swirl the tissue culture plate to ensure thorough mixing. Exchange the cell culture medium with DMEM complete medium six hours post-infection, and incubate the transfected HEK-293T cells at 37 degrees Celsius with 5% carbon dioxide for 48 hours. Collect the supernatant of the infected HEK-293T cells, which contains the SARS-CoV-2 virus-like particles. Filter the collected supernatant through a 0.45 micrometer syringe filter to remove cellular debris. This is the SC2-VLP medium. Seed 40,000 HEK-293T cells with stable expression of Angiotensin-Converting Enzyme 2, or ACE2, and TMPRSS2 in a 96-well plate, and add 50 microliters of SC2-VLP medium. Incubate the 96-well tissue culture plate at 37 degrees Celsius with 5% carbon dioxide for 24 hours. After incubation, remove the medium from each well of the 96-well plate, and wash once with 100 microliters of PBS prewarmed to 37 degrees celsius. Lyse the HEK-293T cells seeded with ACE2 TMPRSS2 cells with 20 microliters of Passive lysis buffer, and gently rock the sample on an orbital shaker for 15 minutes At room temperature. Spin the 96 well plate at 4,000 G for 15 minutes at four degrees Celsius using a refrigerated microplate centrifuge, and then immediately transfer the plate to an ice bath. Take 100 microliters of reconstituted luciferase assay buffer into a new opaque white 96-well plate, and add 20 microliters of lysate into each well. Mix briefly by pipetting up and down two to three times. Measure the luminescence signal using a plate reader. Next, to assess the composition of the SC2-VLP medium, add 1.36 milliliters of PEG 8000 solution to 10 milliliters of SC2-VLP medium. Place the mixture on an orbital shaker and slowly mix at four degrees Celsius overnight. Centrifuge the solution at four degrees Celsius and 2,000 G for 30 minutes. And collect the SC2-VLP pellet for western blotting analysis. Seed approximately 3 million HEK-293T cells evenly in a glass-bottom culture dish with a 15 millimeter diameter, and allow the cells to adhere and grow until they reach approximately 70% confluency. After transecting the cells as demonstrated earlier with the modified plasmid quantities, gently wash the culture dish twice with one milliliter of ice cold PBS. Add one milliliter of 4% paraform aldehyde fixation solution at room temperature, and incubate for 15 minutes. Wash the cells twice for five minutes each with one milliliter of PBS at room temperature, and permeabilize the cells by adding one milliliter of 0.25% Triton X-100 for 10 minutes. Again, wash the cells twice for five minutes each with one milliliter of PBS at room temperature. Then add one milliliter of 5% bovine serum albumin for one hour to block non-specific antibody interactions. Add approximately 200 microliters of primary antibody solution to cover the glass bottom, and incubate at four degrees celsius overnight. Then, remove the primary antibody solution and wash the cells three times for five minutes each with one milliliter of PBS at room temperature. Now, add a fluorescence-conjugated secondary antibody solution and incubate at room temperature for one hour. After washing the cells three times with PBS, stain the nuclei with 2.5 micrograms per milliliter of Hoechst solution at room temperature for five minutes. Finally, after washing the cells with PBS, observe the S protein or organelle staining before acquiring images using a confocal microscope. This figure illustrates the sensitivity of SC2-VLP production to varying transfection amounts of the plasmid encoding the spike protein. SC2-VLP titer was highest when 0.016 micrograms of S plasmid were transfected and decreased significantly with 0.16 and 1.6 micrograms of s plasmid. The H1271 to E mutation in the spike protein significantly reduced SC2-VLP titer compared to wild-type. The E1262 to H mutation led to a moderate reduction in SC2-VLP titer, while the E1262 to H, H1271 to E double mutation abolished production entirely. Full length S and S-2 protein bands were decreased in the E1262 to H and double mutant lanes, compared to wild-type. S packaging efficiency was also reduced in E1262 to H and H1271 to E mutants and nearly abolished in the double mutant. VLP abundance remained largely unchanged across wild-type and all S mutants. Wild-type S protein colocalized with the cis-Golgi marker GM130, but not with the ER marker Sec61 beta or ERGIC marker ERGIC 53. The H1271 to E mutant S protein displayed diffuse cytoplasmic distribution and lacked colocalization with GM130.
