Recombinant plasmid KMP-11 gene of Leishmania major (pcKMP-11): Production, characterization and sequencing
M. A. Aghei Afshar
A. A. Jafari
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Aim: Kinetoplastid membrane protein-11 expresses life cycle stages of all kinetoplastidae parasites. Previous studies have demonstrated that kinetoplastidae KMP-11 gene is highly conserved and may be useful for vaccine strategies against Leishmaniasis. In this study, we isolated Leishmania major (MRHO/IR/75/ER) KMP-11 gene and formulated a pcKMP-11 recombinant expressing plasmid as a candidate DNA vaccine against cutaneous Leishmaniasis. Methods: After gene amplification, KMP-11 fragments were cloned into pTZ57R/T standard cloning vector and transformed in E. coli, then subcloned into pcDNA3 eukaryotic expression vector and pcKMP-11 recombinant plasmid was transfected to CHO eukaryotic cells. Amplification, sequencing, cloning and transfection of gene were performed successfully. mRNA transcription of KMP-11 gene in CHO cells was confirmed by RT-PCR methods. Results: Sequence results were compared with other records of kmp-11 in gene bank and a 97-99% identity was showed. Comparison of KMP-11 protein with other records showed that this protein have 92 amino acids. Additionally, a silico analysis of 3D structures of the wild type and double mutant KMP-11 proteins show that the mutations in position 16 and 41 have led to a change in structure conformation and stability. Conclusion: Present results show that KMP-11 can be an excellent candidate for immunization against leishmaniasis.