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Cloning and Constructing a Plasmid Encoding Leishmania Eukaryotic Initiation Factor Gene of Leishmania major Fused with Green Fluorescent Protein Gene as a Vaccine Candidate

Issue: 
DOI: 
10.7727/wimj.2014.201
Pages: 
256–9

ABSTRACT

Objective: Leishmaniasis is usually treated by chemotherapy; however, toxicity, resistance and high-cost limits the use of the chemical drugs. Leishmania eukaryotic initiation factor (LeIF) protein acts the same as interleukin (IL)-12 and reduces the secretion of IL-4 in lymph node cells of mice infected with L major. The aim of this study was cloning of the gene encoding LeIF antigen into eukaryotic expression plasmid pEGFP-N1.

Methods: DNA was extracted from Iranian strain of the L major (MRHO/IR/75/ER) promastigotes. The full-length sequence of LeIF was amplified with Pfu DNA polymerase using a specific primer. The amplified LeIF was cloned into a pJET1.2/blunt vector. Then this fragment was digested with HindIII and EcoRI and was subcloned into the pEGFP-N1 vector. Confirmation of the cloning was done by colony polymerase chain reaction (PCR).                                                                                

Results: Leishmania eukaryotic initiation factor gene was successfully cloned and subcloned into pJET1.2 and pEGFP-N1 plasmids, respectively. The results of colony PCR, restriction analysis and sequencing confirmed them.

Conclusion: We cloned LeIF gene which could be expressed in eukaryotic cells in vivo and could be used as a vaccine candidate against leishmaniasis in future studies.

Accepted: 
09 Sep, 2014
PDF Attachment: 
e-Published: 12 May, 2015
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