Generating a Multivalent-Displaying Outer Membrane Vesicle Vaccine
Trascrizione
Perform OMV SpyCatcher purification. Start with centrifuging the bacterial solution. Filter the supernatant with a 0.22-micrometer membrane filter, then concentrate it using a hollow fiber column. Filter the concentrate through a 0.22-micrometer membrane filter, then centrifuge it at 150,000 times g at 4 degrees Celsius for two hours using an ultracentrifuge, and discard the supernatant with a pipette. Resuspend the precipitation with PBS and store it at minus 80 degrees Celsius.
To perform RBD-SpyTag transfection, begin with selecting an appropriate eukaryotic expression system, and culture the cells overnight at 130 rotations per minute at 37 degrees Celsius after recovery. Add 20 microliters HEK293F of cell solution into the automated cell counter. Record the number of cells. Adjust the concentration to 1 times 10 to the sixth cells per milliliter, and then culture the cells at 130 rotations per minute at 37 degrees Celsius for 4 hours.
Filter RBD-SpyTag plasmid through a 0.22-micrometer membrane filter and add 300 micrograms of plasmids into the cell culture medium until the final volume is 10 milliliters. Shake for 10 seconds. Heat PEI to 65 degrees Celsius in the water bath. Mix 0.7 milliliters of PEI with 9.3 milliliters of cell culture medium and shake intermittently for 10 seconds.
Add plasmid solution to the PEI solution. Shake the mixture intermittently for 10 seconds and incubate it at 37 degrees Celsius for 15 minutes. Add the mixture to 280 milliliters of cell culture medium and culture at 130 rotations per minute at 37 degrees Celsius for 5 days.
Next, perform RBD-SpyTag purification. Start with centrifuging the cells at 6,000 times g at 25 degrees Celsius for 20 minutes. and use a pipette to collect the supernatant. Build the column with 2 milliliters of nickel-NTA agarose and wash it three times with PBS.
Add imidazole into the cell supernatant to make the final concentration of 20 millimolar and load the cell supernatant. Add 3 column volumes of PBS containing 20 millimolar of imidazole for washing and collect the washing fraction. Gradient elute with three column volumes of PBS containing low to high concentrations of imidazole. Elute twice for each concentration.
To determine the protein concentration by the BCA method, serially dilute the standard BSA protein solution from 2 to 0.0625 milligrams per milliliter. Dilute the purified OMV SpyCatcher and RBD-SpyTag 10 times. Then, mix BCA working solutions A and B at a ratio of 50 to 1. Add diluted protein solution and mix with BCA working solution. Incubate at 37 degrees Celsius for 30 minutes. Measure the absorbance at 562 nanometers of each well, and calculate the protein concentration from the standard curve.
To perform bioconjugation of OMV SpyCatcher and RBD-SpyTag, mix OMV SpyCatcher in RBD-SpyTag in PBS at a 40 to 1 ratio. Vertically rotate to blend the mixture overnight at 15 rotations per minute at 4 degrees Celsius. Perform purification of OMV RBD using PBS solution using a procedure similar to OMV SpyCatcher purification.
