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