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" Cloning and Expression of gene encoding of Leishmania major TSA. Gilda Eslami "
/گیلدا اسلامی
; Hossein Hejazi, Rasoul Salehi, Ali Khamesipour
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Isfahan University of Medical Sciences
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| Document Type
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Latin Dissertation
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| Language of Document
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English
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| Record Number
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102705
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| Doc. No
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T11287
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QX,70,E76c,2008
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| Main Entry
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Eslami, Gilda
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| Title & Author
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Cloning and Expression of gene encoding of Leishmania major TSA. Gilda Eslami/گیلدا اسلامی
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| College
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Schools, Medical
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| Date
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, 2008
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| Degree
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Medical Parasitology,
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| Page No
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149 p.:ill, diag, tab
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| Note
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Orginal Works
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| Abstract
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Abstract:Background- Leishmaniasis is a complex disease caused by about 20 different species of Leishmania. The disease remained without a defined efficient treatment. Macrophage activation is hallmark of natural leishmaniasis control in vertebrate hosts, and the parasites cope with the oxidative burden, especially peroxides, by tryparedoxin pathway containing tryparedoxin peroxidase (TSA or TRYP). Therefore, TSA is one of the most important molecules in Leishmania viability and then, it may be a good goal for challenging against Leishmaniasis.Objective- In this study, LmTRYP6 gene (Gene Bank Accession No EU251502), a member of tryparedoxin protein family, was cloned in order to keep the construct available for various utilization. Cloning the gene in expression vector as well made the expression and characterization of the concerned protein possible. Methods- L. major (MRHO/IR/75/ER) promastigotes were cultured, DNA and RNA were extracted and the gene of interest was PCR amplified. PCR/ RT-PCR fragments were purified and cloned in pTZ57R/T and pET15b expression vector and the expressed protein was verified using western blot method. Finally, the molecular characteristics of the encoded protein were evaluated using appropriate software tools.Results- The 555bp long gene of interest was successfully cloned in pET15b vector, total protein was extracted and subjected to western blotting. The recombinant protein was verified by anti-his antibody which was clearly demonstrated the expressed protein from cloned gene. Molecular evaluation revealed that the cloned LmTRYP6 gene (GeneBank Accesion No EU251502) encoded a predicted 184 amino acid long protein with a theoretical isoelectric point of 6.1101. BLAST analysis showed a number of changes in amino acid composition including the replacement of conserved Trp 177 in some other tryparedoxin peroxidase by Cys in LmTRYP6 (GeneBank Accesion No ABX26130).Discussion and Conclusion- Based on some evidences, Cys177 in the LmTRYP6 (GeneBank Accesion No ABX26130) is highly reactive and readily participates in catalytic activity of the enzyme Which shows its importance for further studies. To our knowledge this is the first report on LmTRYP6 (GeneBank Accesion No ABX26130) from Iranian isolate of L. major which was used in mass leishmanization and preparation of experimental first generation Leishmania vaccine and leishmaninKey words: Tryparedoxin peroxidase. L. major, peroxiredoxin, TRYP6, Cloning.
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| Descriptor
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1. Leshmania.- Descriptors: Lishmanasis
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Peroxidases
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Leishmanasis
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Protozoan Proteins
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Polymerase Chain Reaction
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RNA
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Hejazi, Hossein, Supervisor
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Salehi, Rasoul, Supervisor
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Khamesipour, Ali, Supervisor
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