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Rivista di Biologia Molecolare e Biotecnologie
Indexed/Abstracted in: EMBASE, Science Citation Index Expanded (SciSearch), Scopus
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Minerva Biotecnologica 2014 September;26(3):175-82
Recombinant plasmid KMP-11 gene of Leishmania major (pcKMP-11): production, characterization and sequencing
Bandani E. 1, Soflaei S. 2, Khalili F. 3, Aghei Afshar M. A. 4, Kooshki H. 3, Abdoli A. 2, 6, Kamali M. 3, Sarvi S. 5, Nasiri V. 2, Jafari A. A. 7, Abkar M. 8
1 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran;
2 Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran;
3 Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran;
4 Department of Nanotechnology, Azad University of Pharmaceutical Sciences, Tehran, Iran;
5 Department of Parasitology, and Mycology Mazandaran University of Medical Sciences, Sari, Iran;
6 Department of Parasitology, Kashan University of Medical Sciences, Kashan, Iran;
7 Young Researchers and Elite Club, Parand Branch, Islamic Azad University, Parand, Iran;
8 Department of Genetics, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran
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.