AbstractLeishmaniasis 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.