Recombinant plasmid KMP-11 gene of Leishmania major (pcKMP-11): Production, characterization and sequencing

Bandani, E. and Soflaei, S. and Khalili, F. and Aghei Afshar, M.A. and Kooshki, H. and Abdoli, A. and Kamali, M. and Sarvi, S. and Nasiri, V. and Jafari, A.A. and Abkar, M. (2014) Recombinant plasmid KMP-11 gene of Leishmania major (pcKMP-11): Production, characterization and sequencing. Minerva Biotecnologica, 26 (3). pp. 175-182.

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Abstract

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.

Item Type: Article
Additional Information: cited By 0
Uncontrolled Keywords: amino acid; complementary DNA; DNA vaccine; kinetoplastid membrane protein 11; messenger RNA; mutant protein; unclassified drug, animal cell; Article; CHO cell line; cloning vector; conformational transition; controlled study; DNA immunization; Escherichia coli; expression vector; gene amplification; gene isolation; gene sequence; genetic conservation; genetic transfection; immunization; kinetoplastid life cycle stage; KMP 11 gene; Leishmania major; microbial gene; molecular cloning; nonhuman; protein stability; recombinant plasmid; reverse transcription polymerase chain reaction; RNA transcription; skin leishmaniasis; wild type, Eukaryota; Kinetoplastida; Leishmania major
Subjects: Parasitology
Divisions: Faculty of Medicine > Basic Sciences > Department of Parasitological & Mycology
Depositing User: editor . 2
Date Deposited: 04 Mar 2017 05:33
Last Modified: 04 Mar 2017 05:33
URI: http://eprints.kaums.ac.ir/id/eprint/565

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