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
چکیده(به انگلیسی؛ چکیدهی فارسی در دسترس نیست)
objective: 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. materials and methods: 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. results: 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