ISESCO JOURNAL of Science and Technology
The Official Journal of ISESCO Centre for Promotion of Scientific Research

Abstract

Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which, in the infected host are obligate intracellular parasite. LACK conserved among related Leishmania species. LACK is the immuno-dominant antigen of L.major which is consi dered as the most promi sing molecule for a recom binant or DNA vaccine against leishmaniasis. MRHO/IR/75//ER (an Ira nian strain) of L.major and LACK gene using PCR method was used for amp lifying of LACK gene. Genomic DNA of LACK protein was extracted and amplificated as a template. Then the PCR product was cloned into PTZ57R/T cloning vector. The PT-LACK recombinant plasmid was extracted from white colonies of E.coli bacteria (TG1 strain) and se quenced. PT-LACK plasmids were digested by Hind III and EcoRI enzymes. Then, the purified digestion pro ducts were ligated to pcDNA3 vector; finally, the pc LACK recombinant plasmid was purified from trans- formed E.coli (TG1 strain) and tranfection of pcTSA recombinant plasmid into the CHO cells then analysis by methods of SDS-PAGE and Western blot. LACK gene has been clo- ned into pcDNA3 cloning vector. Sequence analysis of cloned LACK gene into PTZ57R/T vector high homology 89% with LmjF28.2740 (LACK gene). The recombinant plasmid containing LACK gene was expr essed in CHO cells. The expression of pc-LACK recombinant plasmid was demonstrated by SDS-PAGE and Western-blot. W e cloned LACK gene of L.major in pcDNA3 vectors successfully. Recombinant plasmid was confirmed. Results indicated successful expression of pc-LACK plasmids in eukaryotic cells.

Keywords: Cloning, Expression, Leishmania major, LACK, CHO cells.