Cloning and Expression of Recombinant Plasmid Containing P36/LACK Gene of Leishmania infantum Iranian Strain
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Abstract
Background:There are several methods, such as vaccination, to control visceral leishmaniasis. Although there is no efficient vaccine, it seem DNA vaccination with stimulates both cellular and humoral immunity apparently is the best way. The aim of this study was cloning and expression of LACK gene, a 36kD protein, as a can-didate protein for vaccination against Iranian L.infantum.
Methods:Iranian strain of L. infantum [MCAN/IR/07/Moheb-gh] was used as a template for PCR to amplify LACK gene. The LACK gene was cloned in pTZ57R/T vector and after confirmation it was digested by restriction enzymes (BamH1) and cloned in pcDNA3.1 expression vector. Recombinant plasmid was extracted and analyzed by sequencing, restriction digestion analysis and PCR reac-tion. The pc- LACK recombinant plasmid was purified from transformed E.coli (DH5α) and its expression was analyzed by SDS-PAGE and Western blot.
Results:The results of sequencing, restriction digestion analysis and PCR reaction revealed that LACK gene was cloned correctly in pcDNA3.1 vector and the results of SDS PAGE and Western blot emphasized that LACK protein of Iranian L. in-fantum is a well-expressed protein.
Conclusion:We amplified, cloned and expressed Iranian L. infantum LACK genes successfully.
Iqbal A, Mir I, Hafiz Y, Gazal S. Recent Ad-vances in Canine Visceral Leishmaniasis. In-dian Pet J. 2011; 3.
Moshfea A, Mohebalia M, Edrissiana Gh, Za-rei Z, Akhoundia B, Kazemi B, Jamshidi Sh, Mahmoodi M. Canine visceral leishmaniasis:
Asymptomatic infected dogs as a source of L. infantum infection. Acta Trop. 2009;112: 101–105.
Oliva G, Foglia Manzillo V, Pagano A. Canine leishmaniasis: evolution of the chemotherapeu-tic protocols. Parassitologia. 2004; 46(1-2):231-4.
Mohebali M. Visceral leishmaniasis in Iran: Review of the Epidemiological and Clinical Features. Iran J Parasitol. 2013; 8(3): 348-358.
Mohebali M, Hamzavi Y, Fallah E, Zareii Z. Study of canine visceral leishmaniasis in some parts of Islamic Republi of Iran and its health importance. J Vet Res. 2001; 56(3): 55-59
Mohebali M, Hajjaran H, Hamzavi Y et al. Ep-idemiological aspects of canine visceral leish-maniasis in the Islamic Republic of Iran. Vet Parasitol. 2005; 129: 243–251.
Oliva G, Scalone A, Foglia Manzillo V, Gramicca M, et al. Incidence and Time Course of Leishmania infantum Infections Examined by Parasitological, Serologic, and Nested-PCR Techniques in a Cohort of Naive Dogs Ex-posed to Three Consecutive Transmission Sea-sons. J Clin Microbiol. 2006; 44(4): 1318–1322.
Solano-Gallego L, Koutinas A, Miro G, et al. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmanio-sis. Vet Parasitol. 2009; 165: 1–18.
Gradoni L. An update on antileishmanial vac-cine candidates and prospects for a canine Leishmania vaccine. Vet Parasitol. 2001; 100: 87–103.
Handman E. Leishmaniasis: Current Status of Vaccine Development. Clin Microbiol Rev. 2001; 14(2): 229-243.
Shaddel M, Oormazdi H, Fateminasab F, Farahyar S. Efficacy of different Immunization methods against Leishmania Parasite in order to prophylaxis of Leishmaniasis. JAUMS. 2004; 2:359-364.
Ahmed SBH, Bahloul C, Robbana C, Askri S, Dellagi K. A comparative evaluation of differ-ent DNA vaccine candidates against experi-mental murine leishmaniasis due to L. major. Vaccine. 2004; 22: 1631-1639.
Gómez-Arreaza A, Acosta H, Barros-Álvarez XL et al. Leishmania mexicana: LACK (Leishma-nia homolog of receptors for activated C-ki-nase) is a plasminogen binding protein. Exp Parasitol. 2011; 752–761.
Tapia E, Pérez-Jiménez E, López-Fuertes L et al. The combination of DNA vectors express-ing IL-12 + IL-18 elicits high protective im-mune response against cutaneous leishmaniasis after priming with DNA-p36/LACK and the cytokines, followed by a booster with a vaccinia virus recombinant expressing p36/LACK. Mi-crobes and Infection. 2003; 5: 73–84.
Melby C, Yang P, Zhao J, et al. Leishmania do-novani p36 (LACK) DNA Vaccine Is Highly Immunogenic but Not Protective against Ex-perimental Visceral Leishmaniasis. Infection and Immunity. 2001; 69(8): 4719–4725.
Shirali S, Haddadzadeh HR, Mohebali M, Kazemi B. Cloning and Sequencing of Leishma-nia infantum Iran Strain LACK Gene. J Vet Mi-crobiol. 2013; 9(2): 129-138.
Noli CH. Canine leishmaniasis. Waltham Fo-cus. 1999; 9(2): 18-24.
Evans J, Kedzierski K. Development of Vac-cines against Visceral Leishmaniasis. J Trop Med. 2012; 1-14.
Bolhassani A, Rafati Yazdi S. DNA Immuniza-tion as an Efficient Strategy for Vaccination. Avicenna J Med Biotech. 2009; 1(2): 71-88.
Kelly L, Stetson BB, Locksley DM. Leishmania major LACK Antigen Is Required for Efficient Vertebrate Parasitization. J Experi Med. 2003; 198: 1689–1698.
Gradoni L. Vaccine against Leishmaniasis: State of the art. Vet Res Communi. 2006; 30: 59-63.
Ramos A, Alonsoa JM, Marcen A et al. Heter-ologous prime-boost vaccination with a non-replicative vaccinia recombinant vector ex-pressing LACK confers protection against ca-
nine visceral leishmaniasis with a predominant Th1-specific immune response. Vaccine. 2008; 26: 333-344.
Gonzalo RM, Ramón Rodríguez J, Rodríguez D et al. Protective immune response against cutaneous leishmaniasis by prime/booster im-munization regimens with vaccinia virus re-combinants expressing Leishmania infantum p36/LACK and IL- 12 in combination with purified p36. Microbes and Infection. 2001; 3: 701−711.
Gonzalo RM, Realb GR, Rodrigueza J et al. A heterologous prime–boost regime using DNA and recombinant vaccinia virus expressing the Leishmania infantum P36/LACK antigen pro-tects BALB/c mice from cutaneous leishmani-asis. Vaccine. 2002; 20: 1226–1231.
Ramiro J, Zárate MJ, Hanke J et al. Protection in dogs against visceral leishmaniasis caused by Leishmania infantum is achieved by immuniza-tion with a heterologous prime-boost regime using DNA and vaccinia recombinant vectors expressing LACK. Vaccine. 2003; 21: 2474–2484.
Jorjani ON, Ghafarifar F, Sharifi Z, Dalimi A, Hassan ZM. Cloning and sequencing of L. ma-jor homologue of receptors for activated C ki-nase (LACK) gene. Modares J Med Sci. 2009; 11(3-4):19-30.
Jorjani O, Ghaffarifar F, Sharifi Z, Dalimi A. Cloning and Expression of Recombinant Plas-mid Containing LACK Gene of Leishmania ma-jor (MHRO/IR/75/ER) in CHO Cells. Isesco J Sci Technol. 2012; 8(13): 37-43
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| Issue | Vol 10 No 2 (2015) | |
| Section | Original Article(s) | |
| Keywords | ||
| Cloning Expression LACK Leishmania infantum Visceral leishmaniasis | ||
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