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Malaysian Journal of Medical Sciences
School of Medical Sciences, Universiti Sains Malaysia
ISSN: 1394-195X
Vol. 19, No. 1, 2012, pp. 15-19
Bioline Code: mj12003
Full paper language: English
Document type: Research Article
Document available free of charge

Malaysian Journal of Medical Sciences, Vol. 19, No. 1, 2012, pp. 15-19

 en Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania major check for this species in other resources and Expression in the Chinese Hamster Ovary Cell Line
Ghaffarifar, Fatemeh; Tabatabaie, Fatemeh; Sharifi, Zohreh; Dalimiasl, Abdolhosein; Hassan, Mohammad Zahir & Mahdavi, Mehdi

Abstract

Background: TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major check for this species in other resources and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells.
Methods: Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western blotting.
Results: The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots.
Conclusion: The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.

Keywords
CHO cells, gene expression, genetics, Leishmania major, plasmid, recombinant DNA

 
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Alternative site location: http://www.medic.usm.my/publication/mjms/

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