Construction and testing of the plasmid DNA vaccine. (A) Construction of pCMV-HISA70m2A. At the positions marked “G”, four tandem repeats of GGC, encoding four Gly residues, were inserted to improve structural flexibility of the HISA70 protein. (B) Analysis of HISA70 expression in CHO-K1 cells after transfection. Lane 1, cells transfected with pCMV-HISA70m2A, 148 kDa; lane 2, cells transfected with empty vector (pCMVβ-m2A); lane 3, nuclear extracts from L. major promastigotes, 13 kDa; lane 4, untransfected CHO-K1 cells. Protein expression was detected using specific anti-H2A sera from dogs with leishmaniosis. Proteins were separated on a 10% SDS-PAGE gel. The positions of molecular mass markers are indicated on the left.