Expression of 1B capsid protein of Foot-and-mouth disease virus (FMDV) using baculovirus expression system and its validation in detecting SAT 2- specific antisera

Figures

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  Figure 1. The recombinant cassette, shows coding sequence of the 1A-1B inter-peptide (1A1B-IP is a short polypeptide linker that connects VP4/1A and VP2/1B, the signal peptide (SP), histidine tag, enterokinase recognition sequence and the 1B capsid protein, in fusion with a signal peptide.

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  Figure 2. Construction of pFastBacDual vector charboring the 1B cassette. (A) Plasmid map shows the recombinant cassette that was cloned downstream from the Ppol promoter of the modified pFastBac-Dual vector. (B) A total of 1% agaros gel shows the verified recombinant plasmid using restriction enzyme digestion. M: 1 kb ladder. RP: Recombinant plasmid harboring the 1B cassette and the SipS gene. Arrows show the molecular size (kb) for both SipS gene (0.57 kb) and the pFastBac-1B vector (5.9 kb).

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  Figure 3. PCR detection of recombinant bacmid in infected S9 cells. M. 1kb Ladder, lane 1: Total DNA from Sf9 cells (mock infection), lane 2. Total DNA from infected Sf9 cells, lane 3: pFastBacDual vector harboring the 1B cassette (positive control), lane 4: Total DNA from healthy Sf9 cells (mock infection), lane 5: Total DNA from infected Sf9 cells and lane 6: pFastBacDual vector harboring the 1B cassette (Positive control).

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  Figure 4. Protein analysis of 1B expressed in SF9 cells. (A) SDS-PAGE (12%) of total protein extracted from culture filtrate of infected Sf9 cells using recombinant virus. M: Page Ruler prestained protein ladder. C-: Cells lysate extracted from mock infected Sf9 cells. S: Total protein of cells lysate extracted from viral infected Sf9 cells. P: Total protein extracted from culture filtrate (media) of infected Sf9 cells. (B) Western blot analysis of the expressed 1B capsid protein in Sf9 cells. PW: Secreted proteins in culture filtrate of Sf9 infected cells, C+: Positive control. Arrows show a clear protein band at ∼22 kDa corresponding to 1B capsid protein in both SDS-PAGE gels and western blot.

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  Figure 5. Antigen sensitivity of r1B using indirect ELISA. The sensitivity of the r1B capsid protein against FMDV SAT 2 specific antibodies was tested using ELISA assays. The Y-axis represents the optical density value (OD) measured at 450 nm. The x-axis represents serial dilutions of the Sf9-culture filtrate contain the r1B protein. The error bar represents 5% more or less of the ELISA reading value.