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The present protocol describes the bioengineering of outer membrane vesicles to be a "Plug-and-Display" vaccine platform, including production, purification, bioconjugation, and characterization.

Transcript

Nanoscience can effectively enhance the antigen-specific immune response after displaying antigens on the surface of The main advantage in this vaccine platform can achieve the go off plaque and display. In other words, the platform is able to display the variety of antigens in This method can also be applied to other biological nanoparticles to display engine to nano-sized carriers. To perform cytolysin A spy catheter transformation, start with adding five microliters of plasma solution to 50 microliters of BL 21 competent strain.

Gently blow and let cool on ice for 30 minutes. Place the solution for 90 seconds in the water bath at 42 degrees Celsius, and then immediately put the mixed solution on ice for three minutes. Add 500 microliters of luria bertani medium to the bacterial suspension and after mixing culture at 220 rotations per minute at 37 degrees Celsius for one hour.

Next, plate all the transformation luria bertani agar plate containing ampicillin and culture overnight at 37 degrees Celsius. For OMV spycatcher production, start by isolating a single colony from the plate to 20 milliliters of luria bertani medium and culture overnight at 220 rotations per minute at 37 degrees Celsius. Incubate the bacterial solution into a two liter medium and culture at 220 rotations per minute at 37 degree Celsius for five hours until the logarithmic growth stage is reached.

Add isopropyl beta D-IPTG to make the final concentration of the bacterial solution to be 0.5 millimolar and then culture overnight at 220 rotations per minute at 25 degree Celsius. Next, perform OMV spycatcher purification. Start with centri fusing 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 centri fuse it at 150, 000 times G at four degree Celsius for two hours using an ultra centri fuse, and discard the supernatant with a pipette. Re-suspend 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 HEK 293F of cell solution into the automated cell counter. Record the number of cells, adjust the concentration to one times 10 to the six cells per milliliter.

And then culture the cells at 130 rotations per minute at 37 degrees Celsius for four hours. Filter RBD spy tag 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 five days. Next, perform RBD spytag purification.

Start with centri fusing the cells at 6, 000 times G at 25 degrees Celsius for 20 minutes, and use a pipette to collect the supernatant. Fill the column with two 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 three 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 two to 0.0625 milligrams per milliliter, dilute the purified OMV spycatcher and RBD spy tag 10 times.

Then mix BCA working solutions A and B at a ratio of 50 to one. Add diluted protein solution, and mix with BCA working solution. Incubate at 37 degrees 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 spy tag, mix OMV spycatcher and RBD spytag and PBS at a 40 to one ratio. Vertically rotate to blend the mixture overnight at 15 rotations per minute at four degrees Celsius.

Perform purification of OMV RBD using PBS solution using a procedure similar to OMV spycatcher purification. The plasma design scheme shows that the spycatcher gene is connected with the cly A gene via flexible linker while the spytag connects to the five prime terminal of the RBD gene with a hys tag gene for purification and verification. After ultracentrifugation, almost all the un-reacted RBD spytag remained in the supernatant.

The second ultracentrifugation did not provide a further significant advantage over the first time. The z average hydrodynamic diameter of OMV spycatcher was 133 nanometers while it was 152.6 nanometers for OMV-RBD. These differences may be because RBD increases OMV particle size after intensive displaying.

The results of TEM are consistent with the particle size distribution obtained from the DLS results. Rotation for blending the mixture is very important. The efficiency of reaction can be much lower without rotation.

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Journal (Bioengineering)

Journal (Bioengineering)

A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain

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