en Original article Original article <p> <strong> Introduction: </strong><i>Leishmania tarentolae </i>(<i>L. tarentolae</i>) is a nonpathogenic species of <i>Leishmania</i> genus that can be used as an expression system to produce immunogenic proteins or epitopes <i>in vivo</i> acting as an efficient and safe recombinant live vector in vaccinology. Cysteine proteases (CPs) are one group of Family C1 peptidases in <i>Leishmania</i> that are important for survival of the parasite within the host and are introduced as potential vaccine candidates. Two types of cysteine proteases, CPA and CPB, play a critical role in<i> Leishmania</i>. Previously, it has indicated that immunization with two genes or recombinant proteins of CPA and CPB individually or fused together with various adjuvant or combined with liposome are able to elicit a protective immune response against <i>L. major</i> in BALB/c mice. <strong>Methods: </strong>In this study, for the first time, two lines of recombinant non-pathogenic <i>Leishmania</i> were generated by transfection of a heterologous triple fusion gene encoding <i>cpa/cpb/egfp</i>. For this purpose, two different expressions approaches were taken episomal expression using rDNA promoter, and integrative expression from rRNA locus of genome. <strong>Results:</strong> After genotype confirmation, the fluorescence intensity was monitored in different phases of parasite growth by both fluorescence microscopy and FACS analysis.Western blot and RT-PCR analysis showed that <i>cpa/cpb/egfp</i> tri-fused genes were specifically expressed in both lines of transgenic parasites. <strong>Discussion:</strong> In this study a single fused gene was expressed in both systems and the results showed that the expression of integrated gene was higher than episomal. In addition, the present data suggest that <i>L. tarentolae</i> expressing <i>cpa/cpb/egfp</i> is a promising carrier as vectored vaccine in future research. <i>Vac Res</i> <i>, 2014, 1 (1): 1-9 </i></p> Leishmania tarentolae, Knock-in, Cysteine Protease, EGFP 1 9 http://vacres.pasteur.ac.ir/browse.php?a_code=A-10-27-9&slc_lang=en&sid=1 Tahereh Taheri No Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran Elham Gholami No Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran Faeze Saatchi No Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran Negar Seyed No Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran Yasaman Taslimi No Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran Sima Rafati s_rafati@yahoo.com Yes Molecular Immunology and Vaccine Research Laboratory, Pasteur Institute of Iran, Tehran, Iran