Co-expression of the flaviviral E and prM proteins have been reported in a variety of animal cell lines to result in the formation of VLPs. These VLPs have been reported to induce neutralizing antibodies against the wild-type virus particles, indicating their potential as possible sub-unit vaccine candidates (14,15). Biophysical methods could be used to determine the presence of VLPs by performing a density gradient, and analyzing each fraction using western blotting with antisera to the specific protein of interest. The morphology and size of the VLPs could also be visualized under the transmission electron microscopy (TEM). The methods used to confirm VLP formation with recombinant DEN E protein in P. pastoris are described under Subheadings 3.4.1.-3.4.4.
3.4.1. Sucrose Gradient Centrifugation
1. Induce the transformant containing the CprME construct (see Subheading 3.2.1., step 8).
2. Prepare a clarified yeast lysate.
3. Prepare a gradient in the centrifuge tube from 5 to 50% of sucrose solutions made up in PBS (see Note 4).
4. Leave the tube overnight at 4°C or 4 h at room temperature.
5. Layer the clarified lysate, containing the CprME proteins over the gradient.
6. Centrifuge at 4°C for 16 h at 100,000g in the Beckman SW 41 Ti rotor.
8. Analyze each fraction with Western blotting with polyclonal antibody to E protein (see Subheading 3.2.1., steps 9 and 10), and analysis by transmission electron microscopy (see Subheading 3.4.2.).
3.4.2. Electron Microscopy Analysis
1. Place 10 ^L of each sucrose fraction onto the EM grid. Drain dry.
2. Wash in PBS, pH 7.4 for 10 min at room temperature.
3. Fix in 2% glutaraldehyde for 5 min at room temperature.
4. Wash in PBS, pH 7.4 for 5 min at room temperature. Repeat four times.
5. Wash grid in distilled water for 5 min at room temperature. Repeat four times.
6. Stain suspension with uranyl acetate for 5 min at room temperature.
8. Examine with a transmission electron microscope.
3.4.3. Immunoflorescence Assay
1. Fix virus-infected Vero cells in prechilled acetone for 10 min.
2. Incubate the virus-infected cells with preimmune and immune sera for 1 h at 37°C.
3. Wash cells three times with PBS.
4. Stain with donkey anti-rabbit IgG conjugated to FITC, and incubate further for 1 h at 37°C.
5. Wash cells three times with PBS.
6. Mount cells with florescent mounting fluid, and examine with florescence microscopy.
To determine whether the co-expression of C, prM, and E could form VLPs, the yeast transformmant containing the CprME construct was induced, the cells lysed, and the clarified lysate applied to a 5 to 50% sucrose gradient, which was centrifuged for 16 h at 100,000g (Fig. 5A). Our results indicate that fraction 3, collected from the top of the gradient, indicates the presence of E protein when analysed by Western blotting with the antiserum raised to bacterial-expressed E protein (lane 3). When the peak fraction 3, was further analyzed by TEM, VLPs were observed at a high magnification of X60,000 (Fig. 5B).
The immunogenic nature of the VLPs was further examined by immunizing rabbits with the purified VLPs harvested from fraction 3 (six doses of approx 25 ^g per dose were administered to each rabbit under standard procedures). DEN 1 virus-infected Vero cells showed positive florescence staining when reacted with the antisera raised against the VLPS fraction, but not with the preimmune serum (Fig. 6).
A Tia 345673 3 10 11 12 13 P
Fig. 5. Biophysical analysis of recombinant E proteins. Protein expression in the yeast transformant containing the CprME construct was induced, the cells lysed and the clarified lysate applied to a 5 to 50% sucrose gradient. (A) The sucrose gradients were fractionated and the individual fractions analyzed by Western blotting with E protein antiserum. Lane T represents the total sample applied to the gradient before centrifugation, 1 to 13 fractions collected from the top of the gradient to the bottom, and P, the pellet obtained after the centrifugation. The arrow indicates the E protein. (B) The peak fraction, 3, was further analyzed by TEM, at a high magnification of X60,000. Bar represents 50 nm.
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