it is a zoonotic endemic disease in most of the mediterranean area , asia , and latin america ( 2 , 3 ) . in iran the prevalence of cvl in various parts of iran is different and it is correlated by weather condition and humidity ( 4 ) . it is endemic in ardebil , east azerbaijan , fars and bushehr provinces ( 5 ) . domestic dogs ( canis familiaris ) are the main reservoir hosts of human visceral leishmaniasis in iran ( 6 ) and the prevalence of vl in human , in distinct area , is associated with amount of cvl in dogs ( 4 ) . in foci of canine leishmaniasis , symptomatic disease is low and due to the systemic nature of the disease , clinical manifestations are variable ( 68 ) . it seemed among different control strategies of the disease vaccination , if an efficient vaccine being accessible , is the best possible way for eradication of this disease in dogs ( 6 ) . immunization with naked dna is the latest method , which promote both cd4- and cd8-mediated responses ( 9 , 10 ) . in the past few years , studies focused on some antigens such as gp63 , cp , tsa , gp64 , lmsti1 , leif and p8 , p4 and lack ( 11 ) . among of these antigens , lack gene is one of the best candidates in other strains of this pathogen ( 12 ) lack ( leishmania homologue of receptors for activated c kinase ) is a 36 kda protein localized in cytosol and external surface of the membrane ( 13 ) . it is expressed in both promastigote and amastigote forms of the parasite ( 14 ) . the protective effect of the lack vaccine was mediated by il-12-dependent ifn - g production ( 15 ) . the objective of this study was to investigate of the quality of lack protein expression of iran strain of l. infantum . leishmania infantum iran strain [ mcan / ir/07/moheb - gh ] was provided by the school of public health tehran university of medical sciences . promastigotes were grown in rpmi 1640 medium ( gibco , germany ) supplemented with 5% fetal calf serum and 200iu /ml penicillin g [ crystalizeh ] . dna extracted by dna extraction kit - mbst - iran and electrophoresed on 1% agarose gel . the extracted dna was used as a template to amplify the lack gene by pcr . the reaction was performed in 100l of the solution containing : 1l of template dna , 2l of dntp ( 100 mol ) , 0.5l of taq dna polymerase ( 5u/l ) , 10l of 10x pcr buffer , 2 l of mgcl2 ( 50 mmol ) , 79.5l of distilled water and 2l of each of primers ( 10 pmol/l).we designed a pair of primer based on lack gene sequence ( accession number u49695 ) with bamhi and hindii restriction enzymes on 5 forward and reverse primer lack f 5- ggatcc a tga act acg ag - g gtc acc -3 reverse primer : introduced hindiii . pcr product was purified ( purification kit mbst - iran ) , cloned into ptz57r / t vector ( fermentase t / a vector cat . / t - lack was digeted by bamhi and released lack gene purified and sub cloned into pcdna3.1 shuttel vector . briefly , the reaction was performed in 10 l of the solution containing : 5 l of the purified lack gene , 2 l of 5x buffer , 0.5 l of t4 dna ligase ( 5u/l ) ( fermentase ) , 1.5 l of d.w ( distilled - water ) in 4c for overnight and transformed in to competent cell as previously described ( 16 ) . analysis of recombinant colony was done by three methods : colony pcr reaction , restriction analysis and sequencing . the universal primers are used for detection are t7promoter , taa tac gac tca cta tag gc and bgh - rev , cta gaa ggc aca gtc gag gc . a single colony of pc - lack was cultured , when its od reach to 0.6 was induced by 0.1 molar iptg . electrophoresed proteins were transferred to nitrocellulose membrane and western blot analysis was done and specific protein was detected by leishmania antibody - positive dog serum and anti - dog conjugate ( anti - igg dog sigma  ) . for dilution of serum in western blot , the best antigen - antibody reaction was found by dot blot . leishmania infantum iran strain [ mcan / ir/07/moheb - gh ] was provided by the school of public health tehran university of medical sciences . promastigotes were grown in rpmi 1640 medium ( gibco , germany ) supplemented with 5% fetal calf serum and 200iu /ml penicillin g [ crystalizeh ] . dna extracted by dna extraction kit - mbst - iran and electrophoresed on 1% agarose gel . the extracted dna was used as a template to amplify the lack gene by pcr . the reaction was performed in 100l of the solution containing : 1l of template dna , 2l of dntp ( 100 mol ) , 0.5l of taq dna polymerase ( 5u/l ) , 10l of 10x pcr buffer , 2 l of mgcl2 ( 50 mmol ) , 79.5l of distilled water and 2l of each of primers ( 10 pmol/l).we designed a pair of primer based on lack gene sequence ( accession number u49695 ) with bamhi and hindii restriction enzymes on 5 forward and reverse primer lack f 5- ggatcc a tga act acg ag - g gtc acc -3 reverse primer : introduced hindiii . pcr product was purified ( purification kit mbst - iran ) , cloned into ptz57r / t vector ( fermentase t / a vector cat . recombinant ptz57r / t - lack was digeted by bamhi and released lack gene purified and sub cloned into pcdna3.1 shuttel vector . briefly , the reaction was performed in 10 l of the solution containing : 5 l of the purified lack gene , 2 l of 5x buffer , 0.5 l of t4 dna ligase ( 5u/l ) ( fermentase ) , 1.5 l of d.w ( distilled - water ) in 4c for overnight and transformed in to competent cell as previously described ( 16 ) . analysis of recombinant colony was done by three methods : colony pcr reaction , restriction analysis and sequencing . the universal primers are used for detection are t7promoter , taa tac gac tca cta tag gc and bgh - rev , cta gaa ggc aca gtc gag gc . a single colony of pc - lack was cultured , when its od reach to 0.6 was induced by 0.1 molar iptg . electrophoresed proteins were transferred to nitrocellulose membrane and western blot analysis was done and specific protein was detected by leishmania antibody - positive dog serum and anti - dog conjugate ( anti - igg dog sigma  ) . specific binding was revealed with diaminobenzidine ( dab ) ( dako , denmark ) . for dilution of serum in western blot , leishmania infantum genomic dna was extracted and lack gene amplified by pcr reaction . then gene was cloned into ptz57r / t vector and after confirmation plasmid by colony pcr and restriction analysis , recombinant plasmid was digested by bamhi and sub cloned into pcdna3.1 expressing vector ( fig . 3 ) . electrophoresis of t / a - lack and pc- lack recombinant plasmids and pcdna3 plasmid were loaded on a 1% agarose gel./ column1 : t / a - lack recombinant plasmid / column2 : pc- lack recombinant plasmid / column3 : the band of pcdna3 electrophoresis of pcr , amplification of lack gene / with pc - lack./ column 1 : dna ladder / column 27 : pcr amplification of lack gene with pc - lack / column 8 : control negative electrophoresis of extracted pc - lack after digestion by enzyme/ column 1 : dna ladder / column 2 : pclack digested by bamh1 recombinant pcdna3.1 plasmid was purified and sequenced by dideoxy chain termination method and compared with lack gene ( accession no u49695.1 ) by 
 www.ncbi.nlm.nih.gov/blast-showed 
 high homology of 98 % ( fig . 4 ) . sequencing of lack gene with universal primers of pc - dna3.1 a 36 kd band was recognized by leishmania antibody - positive [ polyclonal ] dog sera in protein extracts of the cells transfected with pc - lack . in western blot lack protein ( fig . 5 , 6 ) sds - page analysis of expressed gene product 
 column1:dh5 before expressing by iptg ( control)column2 : pc - dna3.1 before expressing by iptg ( control)column3 : pc - lack before expressing by iptg ( control)/column4 : pre stain protein laddercolumn5 : pc - lack 1h after expressing by iptgcolumn6 : pc - lack 2h after expressing by iptgcolumn7 : pc - lack 3h after expressing by iptgcolumn8 : pc - lack 4h after expressing by iptgcolumn9 : pc - lack 5h after expressing by iptg column1:dh5 before expressing by iptg ( control ) column2 : pc - dna3.1 before expressing by iptg ( control ) column3 : pc - lack before expressing by iptg ( control)/ column4 : pre stain protein ladder column5 : pc - lack 1h after expressing by iptg column6 : pc - lack 2h after expressing by iptg column7 : pc - lack 3h after expressing by iptg column8 : pc - lack 4h after expressing by iptg column9 : pc - lack 5h after expressing by iptg western blot analysis of expressed gene product 
 column1:dh5 before expressing by iptg ( control)column2 : pc - dna3.1 before expressing by iptg ( control)column3 : pc - lack before expressing by iptg ( control)/column4 : pre stain protein laddercolumn5 : pc - lack 1h after expressing by iptgcolumn6 : pc - lack 2h after expressing by iptgcolumn7 : pc - lack 3h after expressing by iptgcolumn8 : pc - lack 4h after expressing by iptgcolumn9 : pc - lack 5h after expressing by iptg column1:dh5 before expressing by iptg ( control ) column2 : pc - dna3.1 before expressing by iptg ( control ) column3 : pc - lack before expressing by iptg ( control)/ column4 : pre stain protein ladder column5 : pc - lack 1h after expressing by iptg column6 : pc - lack 2h after expressing by iptg column7 : pc - lack 3h after expressing by iptg column8 : pc - lack 4h after expressing by iptg column9 : pc - lack 5h after expressing by iptg leishmania infantum genomic dna was extracted and lack gene amplified by pcr reaction . then gene was cloned into ptz57r / t vector and after confirmation plasmid by colony pcr and restriction analysis , recombinant plasmid was digested by bamhi and sub cloned into pcdna3.1 expressing vector ( fig . 3 ) . electrophoresis of t / a - lack and pc- lack recombinant plasmids and pcdna3 plasmid were loaded on a 1% agarose gel./ column1 : t / a - lack recombinant plasmid / column2 : pc- lack recombinant plasmid / column3 : the band of pcdna3 electrophoresis of pcr , amplification of lack gene / with pc - lack./ column 1 : dna ladder / column 27 : pcr amplification of lack gene with pc - lack / column 8 : control negative electrophoresis of extracted pc - lack after digestion by enzyme/ column 1 : dna ladder / column 2 : pclack digested by bamh1 recombinant pcdna3.1 plasmid was purified and sequenced by dideoxy chain termination method and compared with lack gene ( accession no u49695.1 ) by 
 www.ncbi.nlm.nih.gov/blast-showed 
 high homology of 98 % ( fig . a 36 kd band was recognized by leishmania antibody - positive [ polyclonal ] dog sera in protein extracts of the cells transfected with pc - lack . in western blot lack protein ( fig . 5 , 6 ) sds - page analysis of expressed gene product 
 column1:dh5 before expressing by iptg ( control)column2 : pc - dna3.1 before expressing by iptg ( control)column3 : pc - lack before expressing by iptg ( control)/column4 : pre stain protein laddercolumn5 : pc - lack 1h after expressing by iptgcolumn6 : pc - lack 2h after expressing by iptgcolumn7 : pc - lack 3h after expressing by iptgcolumn8 : pc - lack 4h after expressing by iptgcolumn9 : pc - lack 5h after expressing by iptg column1:dh5 before expressing by iptg ( control ) column2 : pc - dna3.1 before expressing by iptg ( control ) column3 : pc - lack before expressing by iptg ( control)/ column4 : pre stain protein ladder column5 : pc - lack 1h after expressing by iptg column6 : pc - lack 2h after expressing by iptg column7 : pc - lack 3h after expressing by iptg column8 : pc - lack 4h after expressing by iptg column9 : pc - lack 5h after expressing by iptg western blot analysis of expressed gene product 
 column1:dh5 before expressing by iptg ( control)column2 : pc - dna3.1 before expressing by iptg ( control)column3 : pc - lack before expressing by iptg ( control)/column4 : pre stain protein laddercolumn5 : pc - lack 1h after expressing by iptgcolumn6 : pc - lack 2h after expressing by iptgcolumn7 : pc - lack 3h after expressing by iptgcolumn8 : pc - lack 4h after expressing by iptgcolumn9 : pc - lack 5h after expressing by iptg column1:dh5 before expressing by iptg ( control ) column2 : pc - dna3.1 before expressing by iptg ( control ) column3 : pc - lack before expressing by iptg ( control)/ column4 : pre stain protein ladder column5 : pc - lack 1h after expressing by iptg column6 : pc - lack 2h after expressing by iptg column7 : pc - lack 3h after expressing by iptg column8 : pc - lack 4h after expressing by iptg column9 : pc - lack 5h after expressing by iptg canine s family , particularly domestic dogs are considered the main reservoir of zoonotic transmission ( 17 ) . dogs usually develop the systemic form of infection , with a highly variable clinical appearance that may involve any organ , tissue , or body fluid and is manifested by non - specific clinical signs . both symptomatic and asymptomatic leishmania - infected dogs act as a source of parasites for vl transmission ( 17 ) . although anti leishmania drugs successfully used for human , vl therapy show low efficacy in canines ( 9 , 18 ) . thus , recent studies have tended to cvl control instead of treating . control methods are variable , such as reservoir control , vector control , insecticide - impregnated materials and culling . but none of them were useful ( 8 , 9 , 19 ) . due to the simple nature of the parasite and this fact that recovery and resistance are the results of reinfection in leishmaniasis , vaccination against vl is feasible ( 13 , 20 ) . studies on a protective vaccine candidate have advanced in recent years and several vaccination methods and several antigens were tested . immunization with naked dna ( dna vaccination ) is a new approach that promote both cd4 and cd8 mediated responses and helped to inducing a protective response against infection ( 10 , 15 , 21 , 22 ) . . found that lack is required for parasite viability and it can be a potential drug target for leishmaniasis ( 20 ) . lack might bind and enhance plasminogen activation in vivo promoting the formation of plasmin and might contribute to the invasiveness of the parasite ( 13 ) . in some studies , for example , the study on the combination of dna vectors expressing il-12 + il-18 and a booster with a vaccinia virus recombinant expressing lack in mice revealed that this combined prime / booster immunization regime is an efficient approach to protect against leishmaniasis ( 14 ) . heterologous prime - boost regime using dna and recombinant vaccinia virus vectors expressing lack was tested in some studies and relative protection was achieved ( 2225 ) . lack dna vaccine induced a robust parasite - specific th1 immune response ( ifn- ) but it is not protective against cutaneous or systemic l. donovani challenge ( 15 ) . cloning and expression of the lack gene of l. major iranian strain for immunology study and production of recombinant vaccine was done before ( 26 , 27 ) . according to prevalence and importance of visceral leishmaniasis in iran and necessity of production an efficient vaccine , we did this study . the results of the blast in gene bank and western blot analyze showed that the gene correctly cloned in vector and it is active in immunological aspect . therefore , this study is a start of design a recombinant vaccine against canine visceral leishmaniasis in the future .