Design, Construction and Immunogenicity Assessment of pEGFP-N1-KMP11-GP96 (Fusion) as a DNA Vaccine Candidate against Leishmania major Infection in BALB/c Mice

Background: kmp-11 (kinetoplastid membrane protein-π) exists in all species of kinetoplastid family. it is fully conserved and the protein produced by this gene can induce a very high cellular immune response. we aimed to design a suitable construction for a leishmania major dna vaccine and evaluate the protective efficacy of it as a candidate for dna vaccine against cutaneous leishmaniasis in balb/c mice. methods: this experimental study was conducted in tehran city, iran, between april 20, 2015 and may 30, 2016. kmp-11 gene of l. major (mrho/ir/75/er, iranian strain) and nt-gp96 of xenopus gp96 dna from a pbluescript-gp96 plasmid were amplified by pcr and the purified pcr products were cloned into the pjet1.2/blunt plasmid vector, then, subcloned into pegfp-n1 plasmid as an expression vector. finally, the kmp-11 gene was fused with gp96 and afterward the combination cloned in pegfp-n1. all the cloned genes confirmed by enzyme digestions. then, four groups of mice were immunized with pbs, pegfp-n1, pegfp-n1-kmp, and pegfp-n1-fusion. four weeks after immunization, all animals were challenged with l. major virulent promastigotes. results: the constructed fusion potentially showed an ability to elicit th1 responses that led to cutaneous lesion healing. interestingly, the group received kmp11-gp96 –gfp showed the highest ratio of ifn- γ /il-4 and igg2a/igg1 compare to other groups. no side effect was observed after using the fusion in the mice. conclusion: the constructed fusion could well stimulate both the cellular and humoral immune systems that led to cutaneous lesion healing in mice.