1 2 3 4 5 6
B
1 2 3 5’ UTR 5’ UTR +C 3’UTR 5’ UTR 5’ UTR +C 3’UTR A
5’ UTR 5’ UTR +C 3’UTR
1 2 3 4 5 6
B
1 2 3 5’ UTR 5’ UTR +C 3’UTR 5’ UTR 5’ UTR +C 3’UTR
10 15 37 75 50 150 20
3.5 RNA pull-down assay
To complement the study of Northwestern blot assay in the study of C and RNA interaction, a RNA pull-down assay was developed. In order to determine the minimum amount of RNA needed to pull-down C protein, the amount of C protein and streptavidin beads was kept constant but a range of concentration was used for the RNA and it was found that as a little as 1.25 ng of RNA could be used to pull down C protein (Fig 3- 11A). Next, the minimum concentration of streptavidin beads needed to pull down the C – RNA complex was determined. The complex was allowed to form and a range of streptavadin concentration was used. It was found that at least 20 µg was needed to pull down detectable amounts of C protein (Fig 3-11B). For subsequent experiments we would be using at least 20 µg of streptavidin beads and at least 0.5 ng of RNA since the RNA can be synthesized in abundance.
Figure 3-11. RNA pull-down with streptavidin magnetic beads. (A) His-C protein was
allowed to complex with 20 ng/ml to 0.5 ng/ml of viral 3’ UTR RNA (Lanes 1-7) and the complex was pulled down with 70 µg of streptavidin beads. (B) His-C protein was allowed to complex with 0.5 ng/ml of viral 3’UTR RNA and the complex is pulled down with a range of concentration of the streptavidin beads. They range from 70 µg to 10 µg (Lanes 1-7). Arrowheads indicate positive pulldown results.
A 1 2 3 4 5 6 7 1 2 3 4 5 6 7 20 15 10 0.5 2.5 1.25 0.5 RNA Concentration (ng/ml) B 1 2 3 4 5 6 7 70 60 50 40 30 20 10 Streptavidin beads (µg)
3.6 Production and validation of anti-C antibodies with His-C protein
Twenty-five µg of His-C protein was injected into the mouse every fortnight and the mouse was sacrificed 8 weeks after the first injection. The serum was harvested and tested. An immunoblot was performed to test the sensitivity of the serum to detect His-C protein derived from infected cell lysate and C protein peptide fragments (Fig. 3-12). In addition to immunoblot assays, the antibodies were tested on infected cells and visualized with fluorescence microscopy (Fig. 3-13).
Figure 3-12. Validation of serum from mouse inoculated with C protein via immunoblot.
(A) His-C protein (Lane 1), mock-infected BHK cell lysate (Lane 2) and infected-BHK cell lysate (Lane 3) were blotted onto a nitrocellulose membrane after SDS-PAGE. The membrane was incubated with inoculated mouse serum diluted 1:100 in 5 % milk in TBST. The antibody bound to the membrane was then detected using anti-mouse antibody conjugated to HRP. Finally the blot was developed with a chemiluminescent assay. The antibody was able to detect His-C protein (arrow) as well as the dimeric form (small arrow) of the C protein in infected cells. (B) Overlapping peptide fragments spanning the entire C protein (Lanes 1-6) were synthesized and dot blotted onto a nitrocellulose membrane. The membrane was incubated with mouse serum diluted 1:100 in 5 % milk in TBST and the anti-C antibody bound to the membrane was then detected using anti-mouse antibody conjugated to HRP. Finally the blot was developed with a chemiluminescent assay. Only peptides 1-5 react with the antibody since peptide 6 was not expressed in the His-C protein
B 1 2 3 4 5 6 1 2 3 4 5 6 1-23 18-45 41-64 61-84 79-105 100-123 Peptide Fragment A His-C Mock in fe ct ed B H K c el l ly sa te In fe ct ed B H K c el l l ysa te 1 2 3 Dimeric His-C Monomeric His-C
Figure 3-13. Immuno-staining of WNV C protein in infected cells with anti-C antibodies.
BHK cells is mock-infected (A) or infected with WNV virus and fixed at 6 hr (B) or 24 hr (C) post-infection. The cells were then probed with anti-C antibodies to test the specificity and sensitivity of the antibodies. Anti-mouse alexafluor 594 secondary antibodies were then used to stain the cells. The nuclei of the cells are stained with DAPI. C proteins are stained predominately in the nuclei at 6 hr post-infection (B) while the C proteins are stained predominately in the cytoplasm at 24 hr post-infection (C). The scale bars are shown on the bottom right corner.
C A A Mock-infected B 6 hr post-infection 24 hr post-infection C