Figure 1.
The IAPV genome and origin of sequences used for IAPV fragment expression.
A. Genome structure of IAPV dicistrovirus with the two open reading frames and their constituent mature proteins shown. IRES – internal ribosome entry site, IGR – intergenic region. B. The fragments targeted for expression in E.coli and their encoded products. The precise endpoints used were: VP2, VTH→MQC; VP2-4, VTH→FGW; VP3, SKP→ELQ; VP1, INI→ISR. Expression screening for the proteins predicted from the fragments shown in B. C- left. Western blot with anti-His antibody. C-right. Purified VP2 used for the generation of a rabbit serum. Numbers to the left of the blot are protein molecular mass markers and are in kilodaltons.
Figure 2.
Design and test of baculovirus expression cassettes.
A. Cartoon representations of the various genetic constructs used to assess IAPV antigen expression in infected Sf9 cells. The sequences used are identified. FS- the HIV-1 frameshift site, the polypyrimidine tract of which is indicated. 3C-pro – the IAPV 3C like protease. B. Western blots of Sf9 cells infected for 2 days with recombinant baculoviruses constructed with the cassettes shown in A. In B the upper panel was blotted with rabbit anti-VP2 serum, the middle panel with anti-gp64 and the lower panel is the relative VP2:gp64 level. Numbers to the left of the blots are protein molecular mass markers (M) and are in kilodaltons. The expected position of the blotted antigens is indicated.
Figure 3.
Immunofluorescence of Sf9 cells infected for 2 days with constructs ORF2 and ORF2-5TFS-3C stained with anti IAPV VP2 and an anti-rabbit Alexa Fluor 488 conjugate.
A – Sf9 mock infected control. B – Sf9 cells infected with construct ORF2 expressing ORF2 only. C – Sf9 cells infected with construct ORF2-5TFS-3C expressing ORF2 fused with an attenuated 3C like protease. Occasional punctate staining is apparent in C.
Figure 4.
Analysis of empty IAPV capsids.
A. Sucrose gradient analysis of Sf9 cell extracts infected with recombinant baculovirus ORF2-5TFS-3C. Each fraction represents a 5% step from 30% to 60% sucrose w/v and is blotted with the IAPV anti VP2 serum. B. EM analysis of peak fractions from the sucrose gradient shown in A. Empty capsids are indicated. A damaged particle is labelled D. Baculovirus nucleocapsids are labelled B. The bar is 100 nm.
Figure 5.
Development of an IAPV capture ELISA.
A. Pepscan of 4 monoclonal antibodies to IAPV VP2 on an overlapping peptide library. Filled bars IAPVMAb8; stipple bars IAPVMAb12; striped bars IAPVMAb17; open bars IAPVMAb 27. The relationship between peptide identity and VP2 structure is shown. B. Twin site capture ELISA using capture MAbs 8 (triangles) and 27 (diamonds) as capture layer and probing with HRP-labelled IAPV MAb12. The test sample was a lysate of Sf9 cell infected with the baculovirus expressing ORF-2-5TFS-3C.