[Construction of pcDNA3.1+ vector containing FMDV type O/IRN/1/2007-VP1 gene, confirmation of protein expression in BHKT7 cells and evaluation of immune response in mice model]

ABSTRACT

The aim of this study was to construct a pcDNA3.1+vector containing FMDV type O/IRN/1/2007-VP1 gene, protein expression in BHKT7 cells and evaluation of immune response in BALB/c mice. FMDV type O/IRN/1/2007 was isolated from a cattle in Ray in 2007 and serotyped. The purified VP1 gene was sub-cloned into the PTZ57R/T vector and pcDNA3.1+expression vector. The PCR product of Vp1 gene without stop codon was sub-cloned upstream of EGFP gene into the pEGFP-N1 vector to evaluate VP1-GFP fusion protein expression. The pcDNA3.1-VP1 and pEGFP-VP1 vectors were transfected into BHKT7 cell line. The expression of VP1 protein was evaluated by SDS-PAGE, western blotting and florescent analysis of VP1-GFP fusion protein. The mice were injected subcutaneously by pcDNA3.1-VP1 vector as DNA vaccine and titration of neutralizing antiserum and T cell proliferation assay were done to evaluate the immune response. Insertion of VP1 gene was confirmed by double digestion of sub-cloned PTZ57R/T, pcDNA3.1+and pEGFP-N1 vectors. The specific band in western blotting was also confirmed the VP1 protein expression in BHKT7 cells. The expression of VP1-GFP fusion protein was observed under the immune-florescent inverted microscopy as more green florescent spots versus expression of GFP protein, alone. The neutralizing antiserum titer and T cell proliferation increased significantly in the group of mice vaccinated with pcDNA3.1+-VP1 vector verses control groups [P<0.05]. The results showed that the target gene was amplified, cloned in the cloning and expression vectors and protein expression was confirmed successfully. According to the confirmed VP1 protein expression and increasing neutralizing antiserum titer and T cell proliferation by pcDNA3.1+-VP1 vector [P<0.05], it can be used as DNA vaccine against FMDV type O/IRN/2007