Designing and making the molecular construct for the production of Leishmania major recombinant antigens using pLEXSY-neo2.1 vector

Introduction and Objectives: The annual incidence of leishmaniasis is increasing and there are some active centers of this disease in different geographic locations of Iran. Yet, no effective vaccine has been made for human. One type of vaccine is a recombinant subunit vaccine consist immunogenic antigens of this parasite. The aim of this study was design and develop of the molecular construct using pLEXSY-neo2.1 vector to produce LACK and KMP11 antigens of L. major in L. tarentulae. Materials and Method: The sequences of genes together GFP gene were synthesized within the pLEXSY-neo 2.1 vector. For LACK gene, utr1 vector pLEXSY was used for Trans splicing. Upstream sequences UTR of L. donuani alpha-tubulin gene was used for polyadenylation of the LACK gene and trans splicing of the KMP11 gene. As well as, downstream sequences UTR of the alpha-tubulin L. donuni were used for the polyadenylation of the KMP11 gene and trans-splicing EGFP gene. The LACK and KMP11 proteins were designed as a secretion. Then, the peptide signal sequence for these two genes was placed within construct. To extract these two proteins after expression, the His-tag sequence for the LACK protein and the S-tag sequence for the KMP11 protein were placed in construct. The vector pLEXSY-neo2.1 / LACK-KMP11-EGFP was cloned in E. coli strain Top10 strain. After linearization with the SwaI enzyme, transfected into the L. tarentulae by electroporation. Results: After selection of recombinant strains by neomycin, were confirmed by PCR. Recombinant proteins were extracted and purified from the culture medium and approved by Western blot method. Discussion: L. tarentulae is a non-pathogenic parasite that considered as a microorganism producing various eukaryotic proteins, recently. In this study, designed structures capable to produce antigens in this parasite. Due to the convenience and low cost of cultivating this microorganism, as well as the significant amount of production proteins and similarity of the epitopes produced by the antigens Leishmania, this parasite can be used to develop and produce recombinant vaccines. Acknowledgments: The authors would like to thank the National Institute for Medical Research Development (NIMAD), for financial support (Grant number: 957777).

Keywords:

L. tarentula , L. major , recombinant antigens , pLEXSY-neo2.1 Vector