The overall goal of this procedure is to study virus assembly in vivo and in vitro. This is accomplished by first expressing bro mosaic virus RNAs and bacteria, and exposing it to plant cells through agro infiltration. Next, the BMV virions are purified from infiltrated leaves.
Then capsid protein subunits for in vitro assembly are prepared. Finally, BMV Varians are assembled in vitro using the capsid protein and indocyanine green dye. Ultimately, the results show in vitro assembled bro mosaic varians through formation of optical ghosts, which can be determined by absorbance and emission of fluorescence.
So today we're going to show a technique which is referred as filtration, and this technique is useful for delivering viral components to the plants. And the main advantage of this technique is to eliminate making in vitro transcripts, and at the same time, it delivers multiple components to cell to the same cell, which permits a synchronized infection. The work that we are currently pursuing is the use of the bro mosaic virus in combination with an FDA approved chromophore in the cyanide green to fabricate sort of a new type of a nano particle for biomedical applications.
And specifically our intention is to use these constructs to which we refer to as optical wire ghosts or OB gs for optical imaging modalities, including fluorescence imaging and photoacoustic imaging, and also as a vehicle for laser therapy. A specific clinical application that we are currently pursuing is to use these constructs for imaging ovarian cancer in conjunction with intraoperative procedures. And the idea is that we can functionalize these constructs.
The OBG with the specific targeting moti that can recognize residual tumor cells, that in the case of the ovarian cancer that may be especially within the peritoneal cavity From a plate of previously prepared agrobacterium harboring the brown mosaic virus RNA one, two, or three choose a single colony to inoculate two milliliters of LB containing 50 micrograms per milliliter of can mycin and 100 micrograms per milliliter of rifampicin. Incubate the culture for one day at 28 degrees Celsius in an orbital shaker at 250 RPM inoculate, 50 milliliters of lb in a 500 milliliter erlenmeyer flask with one milliliter of culture and incubate for 16 hours at 28 degrees Celsius in an orbital shaker at 250 RPM the following day. When the cultures have reached an OD 600 of 1.0, transfer them to sterile 30 milliliter tubes and centrifuge for 10 minutes.
At 5, 000 RPM and four degrees Celsius dissolve the pellets in 10 milliliters of 10 millimolar magnesium chloride and centrifuge for 10 minutes at 5, 000 RPM and four degrees Celsius. Repeat this step one more time to ensure complete removal of the antibiotic. Next, suspend the pellets in 10 milliliters of buffer containing 10 millimolar MES and 10 millimolar magnesium chloride.
Measure the OD 600 for each culture and adjust to an OD 600 of 0.1 using 10 millimolar magnesium chloride and 10 millimolar MES. Then combine 10 milliliters of each of the three 0.1 OD 600 culture suspensions. Add acetophenone to a final concentration of 100 millimolar.
Mix gently and keep the mixture undisturbed at room temperature for three hours. After the incubation, draw the culture into a one milliliter syringe without a needle. Infiltrate the culture suspension into the a axial side of two to three well expanded leaves of N hamana by gently pressing the syringe to one half of the ab axial surface of each leaf.
Keep the infiltrated plants in the greenhouse at 24 degrees Celsius for three to four days before harvesting the leaves. After purifying the BMV Virions, prepare one liter of one x virus dissociation buffer. Next, prepare dialysis membrane.
According to the method of sambrook, etal, dispense five to 10 milligrams per milliliter of purified virus into a dialysis bag and place the bag into a flask of one x virus. Dissociation buffer. Dialyze the virus for 24 hours at four degrees Celsius while stirring.
Collect the solution from the dialysis bag and centrifuge at 30, 000 RPM for 30 minutes at four degrees Celsius to pellet any unassociated virus particles. Centrifuge the supernatant at 30, 000 RPM for three hours at four degrees Celsius. After preparing RNA transcripts to be reassembled into virion, mix capsid protein subunits and RNA transcripts at a ratio of one to five.
Dispense the mixture into a dialysis bag and properly secure it to avoid any leaks. After preparing one liter of one XRNA assembly buffer, dialyze the reassembly reaction mixture in the buffer at four degrees Celsius for 24 hours the following day, remove the mixture from the dialysis bag and add 1.5 milliliters of RNA assembly buffer next centrifuge again at 30, 000 RPM for three hours at four degrees Celsius. Then remove the supernatant and resuspend the pellet.
Next, estimate the virion concentration at OD 260 nanometers. Then determine the concentration of capsid protein subunits at OD 280 nanometers or with other methods such as the Bradford Assay to determine the integrity of the dissociated capsid protein subunits Perform 12 to 15%SDS page followed by western blotting. Finally, follow the procedure outlined in the written protocol to generate optical viral ghosts or ov cheese.
Shown here is a transmission electron microscopic image of negatively stained BMV Varion purified from end hamana plants agro infiltrated with a mixture of all three wild type BMV agri constructs. This TEM image shows negatively stained indocyanine green containing optical viral ghosts. Here, absorbence and fluorescent spectra for optical viral ghosts are shown Once mastered.
This technique can be performed in five minutes per sample. After watching this video, you should be able to have a good understanding how to encapsulate viral or non-viral component in to capsid protein subunits.
