Construction of pcDNA۳.۱+ vector containing FMDV type O/IRN/۱/۲۰۰۷-VP۱ gene, confirmation of protein expression in BHKT۷ cells and evaluation of immune response in mice model

Objective: The aim of this study was to construct a pcDNA۳.۱+ vector containing FMDV type O/IRN/۱/۲۰۰۷-VP۱ gene, protein expression in BHKT۷ cells and evaluation of immune response in BALB/c mice. Materials and Methods: FMDV type O/IRN/۱/۲۰۰۷ was isolated from a cattle in Ray in ۲۰۰۷ and serotyped. The purified VP۱ gene was sub-cloned into the PTZ۵۷R/T vector and pcDNA۳.۱+ expression vector. The PCR product of Vp۱ gene without stop codon was sub-cloned upstream of EGFP gene into the pEGFP-N۱ vector to evaluate VP۱-GFP fusion protein expression. The pcDNA۳.۱-VP۱ and pEGFP-VP۱ vectors were transfected into BHKT۷ cell line. The expression of VP۱ protein was evaluated by SDS-PAGE, western blotting and florescent analysis of VP۱-GFP fusion protein. The mice were injected subcutaneously by pcDNA۳.۱-VP۱ vector as DNA vaccine and titration of neutralizing antiserum and T cell proliferation assay were done to evaluate the immune response. Results: Insertion of VP۱ gene was confirmed by double digestion of sub-cloned PTZ۵۷R/T, pcDNA۳.۱+ and pEGFP-N۱ vectors. The specific band in western blotting was also confirmed the VP۱ protein expression in BHKT۷ cells. The expression of VP۱-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 pcDNA۳.۱+-VP۱ vector verses control groups (P<۰.۰۵). Conclusion: 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 VP۱ protein expression and increasing neutralizing antiserum titer and T cell proliferation by pcDNA۳.۱+-VP۱ vector (P<۰.۰۵), it can be used as DNA vaccine against FMDV type O/IRN/۲۰۰۷.

کلیدواژه ها:

Foot and Mouth Disease Virus ، Vector ، vaccine ، VP۱ protein ، ویروس تب برفکی ، ناقل ، واکسن ، پروتئین VP۱