plasmodium vivax ( p. vivax ) is the second widespread malaria species that causes disease in human and imposes sizeable socioeconomic burden and public health difficulty on many countries , particularly in asia and south and central america ( 1 ) . although , p. vivax is responsible for more than 50% of malaria morbidity outside sub - saharan africa , but little consideration for control and research has been dedicated to this parasite ( 2 ) . at present , about 2.8 billion people universally are at risk of p. vivax infection and occurrence of the disease is nearly 132391 million cases each year ( 3 ) . in a nutshell , in malaria life cycle , being bit by female anopheles mosquitoes , sporozoites are transferred to human and infection is initiated . within hepatocytes , the sporozoites reproduce and increase in quantity to thousands of merozoites , which attack the red blood cells ( rbcs ) in the next stage . in the infected rbcs , the small ring shape changes through trophozoite to schizont , in which it bursts and liberates more merozoites ( 4 ) . up to now , some problems such as non  cultivable nature of p. vivax , caused limitations in the study of its molecular biology , but genetic diversity and population organization of this parasite have become more clear by sequencing and genetic engineering including cloning methods ( 5 ) . several p. vivax antigens have been proposed as detectable potential candidates ( 6 ) , among which c - terminal region of pvmsp-1 has high expression on the mature merozoites surface as well as mass protein production , that it has been proven to take part in the parasite invasion to the erythrocyte . specific antibodies against msp-1 , have been particularly shown react to the c - terminal region ( 33-kda and 19-kda sub fragments ) ( 79 ) . antigenic variation is one of the limitative agents in antibody detection that could be due to haplotype variations on target gene . it can ultimately affect the protein conformation , affinity of antibody - antigen and serological test results . little information is available regarding the genetic polymorphism of msp-142 among iranian p. vivax population . in this study , in order to assess the nature of iranian p. vivax isolate , molecular diversity of cloned pvmsp-142 
 kda gene was analyzed . in this study , 11 isolates of chabahar district ( sistan and balouchestan province ) that had p. vivax infection symptoms were chosen and sequenced in order to surveying their similarities . one out of 11 mentioned isolates which had highest homology was selected to cloning process . . the parasite genomic dna was extracted from the whole blood by genomic dna extraction kit , dng  plus ( cinna gene , iran , dn8118c ) using the kit manufacturer s guidelines . continuously , genomic dna quantity and quality was controlled by electrophoresis on 0.8% agarose gel and a biophotometer ( ependorf ) at 260 and 280 nm . amplification from 42 kda partial regions of pvmsp-1 gene was done which included 19 kda and 33kda fragments . the primers were designed on the basis of the sequence of pvmsp-142 
 kda gene , ( genbank : accession no : dq907673 ) . the following primers were used for sequencing : msp1.42f ( 25mer ) ( 5-ggatccgaccaagtaacaacgggag-3 ) , msp1.42r ( 25mer ) ( 5-gaattccaaagagtggctcagaacc-3 ) . the pcr was performed in a pcr tube containing : 200 ng ( 1.5l ) extracted dna as template , 20 pmol ( 0.25 l ) of each primer , 7.5 l of pcr master mix 2x that contained taq and 5.5 l of ddh2o . the objective gene was amplified for 30 cycles ( initial denaturation at 96 c for 5 min , 96 c for 30s , 58 c for 30s and 72 c for 1 min and final extension for 20 min ) subsequently , the pcr product was controlled on 1% agarose gel against a standard dna ladder ( fermentase co. ) . pcr products that consisted of a single specific band can be directly ligated to t - vectors . however , the removal of the impurities such as primers and nonspecific products by gel separation causes increase in the percentage of colonies containing the correct inserts . thermo - stable dna polymerase which does not preferentially add a 3-a at the ends of the pcr products is essential to removal of that enzyme by gel separation . purification and recovery of the dna from agarose gel will be explained in the next sections . purified dna fragment was inserted into ptz57r / t that provided by the following protocol . any plasmid could be selected to gratifies our requirements including the plasmid contains a unique blunt - end restriction site in the multiple cloning site ( in this study , the ecor v site of the ptz57r plasmid was used that has blue / white color selection ) . digesting plasmids were performed in a 0.5 ml microcentrifuge tube containing : 25 l plasmid dna ( 2g ) , 4 l of 10x ecor v buffer , ( 10 unit ) 1 l of ecor v ( fermentase co. ) and dd h2o up to 40l . then it was mixed by gentle vortex , centrifuged and incubated at 37 c for 2 hours . in the next step , 2 l of the mixture was taken and run on a 1% agarose gel to make sure the digestion was completed . thereafter it was incubated to 65 c by water bath for 10 min at the end of digestion ( fig . a : lane1 : intact plasmid ptz57r , lane2 : digested plasmid ptz57r by ecorv , lane3 : size marker : 1 kb . b : lane1 : recombinant plasmid , lane2 : intact plasmid , lane3 : size marker1 kb . c : lane1 : ( a : separated pcr product from digested recombinant plasmid ) , lane2 : intact recombinant plasmid , lane3 : size marker1 kb taq polymerase and dttp were used to add a 3-t to the blunt - ended plasmid . briefly , the making t - overhangs was performed in a 200 l pcr tube containing : 40 l blunt - ended plasmid dna , 5 l of 10 x pcr buffer ( mgcl2 free ) , 2.5 l of mgcl2 ( 250 mm ) , 0.5 l of dttp ( 100 mm ) , 2 l of taq polymerase ( 5 u/l ) . it was mixed by gentle vortex , centrifuged shortly and incubated at 72 c for 2 h. t - vectors were separated from the self - ligated and concatemerized plasmid dna on a 1% agarose gel by 0.5 g / ml of ethidium bromide . the dna bands were visualized by a hand - held long wave ( 365 nm ) uv light . the gel slice transferred to a 2 ml micro centrifuge tube , and then t - vectors were purified and recovered by silica bead dna gel extraction # k0513 ( fermentas co. ) via the kit manufacturer s guidelines . the density of the purified t - vectors was determined by running 1 l of the t - vector in compared to standard dna ladder in a 1% agarose gel at 5 volts / cm for 45 min and then compared the comparative brilliantness of the bands . 1:3 molar ratio of t - vector to insert dna was recommended , but the amount of a - tailed dna solution in a 10 l ligation should not be more than 2 l . in a sterile 0.5 ml micro centrifuge tube , added : 2 l t - vector , 2 l of a - tailed dna , 1 l of 10 x ligation buffer , 1 l of t4 dna ligase ( 23 u/l ) and 4 l of ddh2o . it was mixed gently , centrifuged briefly and incubated at 14 c for overnight ( fig . before transformation , luria - bertani ( lb ) agar plates contained 100 g / ml of ampicillin , were prepared and spreaded with 5-bromo-4-chloro-3-indolyl--d - galactoside ( x - gal ) and isopropylthio--d - galactoside ( iptg ) . for each ligation reaction , 50 l aliquoted of frozen competent cells ( top 10 ) was thawed on ice and added 2 l of the ligation reaction to the competent cells and mixed gently by stirring with the pipette tip . the cells were incubated on ice for 30 min then heats shocked for 30 s in the 42 c water bath , and then were immediately placed on ice for 2 min . the transformed cells were spreaded on each labeled lb - ampicillin plate with x - gal and iptg . the positive colonies that had white color were identified either by restriction enzyme and screening by pcr method after transformation . pcr screening was carried out with same primers that be used for pcr amplification for insert production . for screening by restriction enzyme method , the insert was released by digestion with two unique restriction enzymes ( with cutting sites , ecor1 : gaattc and bamh1 : ggatcc ) from the multiple cloning sites , and the insert size was confirmed by agarose gel electrophoresis ( fig . pcr product of pvmsp-1 42 kda was directly analyzed on abi prismtm 310 automated sequencer to determine p. vivax variation ( strain / haplotype ) in the infected patient . both directions of our sequences were aligned and edited using sequencher tm 4.0.4 software for pc ( gene codes corporation ) and mega 5 software for phylogenetic analysis and then were compared with some genbank sequences for their similarity ( 10 ) . a phylogenetic hypothesis of p. vivax was generated with pvmsp-1 42 kda using the nonparametric bootstrapping , neighbor joining method . in this study , 11 isolates of chabahar district ( sistan and balouchestan province ) that had p. vivax infection symptoms were chosen and sequenced in order to surveying their similarities . one out of 11 mentioned isolates which had highest homology was selected to cloning process . . the parasite genomic dna was extracted from the whole blood by genomic dna extraction kit , dng  plus ( cinna gene , iran , dn8118c ) using the kit manufacturer s guidelines . continuously , genomic dna quantity and quality was controlled by electrophoresis on 0.8% agarose gel and a biophotometer ( ependorf ) at 260 and 280 nm . amplification from 42 kda partial regions of pvmsp-1 gene was done which included 19 kda and 33kda fragments . the primers were designed on the basis of the sequence of pvmsp-142 
 kda gene , ( genbank : accession no : dq907673 ) . the following primers were used for sequencing : msp1.42f ( 25mer ) ( 5-ggatccgaccaagtaacaacgggag-3 ) , msp1.42r ( 25mer ) ( 5-gaattccaaagagtggctcagaacc-3 ) . the pcr was performed in a pcr tube containing : 200 ng ( 1.5l ) extracted dna as template , 20 pmol ( 0.25 l ) of each primer , 7.5 l of pcr master mix 2x that contained taq and 5.5 l of ddh2o . the objective gene was amplified for 30 cycles ( initial denaturation at 96 c for 5 min , 96 c for 30s , 58 c for 30s and 72 c for 1 min and final extension for 20 min ) subsequently , the pcr product was controlled on 1% agarose gel against a standard dna ladder ( fermentase co. ) . pcr products that consisted of a single specific band can be directly ligated to t - vectors . however , the removal of the impurities such as primers and nonspecific products by gel separation causes increase in the percentage of colonies containing the correct inserts . thermo - stable dna polymerase which does not preferentially add a 3-a at the ends of the pcr products is essential to removal of that enzyme by gel separation . purification and recovery of the dna from agarose gel will be explained in the next sections . purified dna fragment was inserted into ptz57r / t that provided by the following protocol . any plasmid could be selected to gratifies our requirements including the plasmid contains a unique blunt - end restriction site in the multiple cloning site ( in this study , the ecor v site of the ptz57r plasmid was used that has blue / white color selection ) . digesting plasmids were performed in a 0.5 ml microcentrifuge tube containing : 25 l plasmid dna ( 2g ) , 4 l of 10x ecor v buffer , ( 10 unit ) 1 l of ecor v ( fermentase co. ) and dd h2o up to 40l . then it was mixed by gentle vortex , centrifuged and incubated at 37 c for 2 hours . in the next step , 2 l of the mixture was taken and run on a 1% agarose gel to make sure the digestion was completed . thereafter it was incubated to 65 c by water bath for 10 min at the end of digestion ( fig . a : lane1 : intact plasmid ptz57r , lane2 : digested plasmid ptz57r by ecorv , lane3 : size marker : 1 kb . b : lane1 : recombinant plasmid , lane2 : intact plasmid , lane3 : size marker1 kb . c : lane1 : ( a : separated pcr product from digested recombinant plasmid ) , lane2 : intact recombinant plasmid , lane3 : size marker1 kb taq polymerase and dttp were used to add a 3-t to the blunt - ended plasmid . briefly , the making t - overhangs was performed in a 200 l pcr tube containing : 40 l blunt - ended plasmid dna , 5 l of 10 x pcr buffer ( mgcl2 free ) , 2.5 l of mgcl2 ( 250 mm ) , 0.5 l of dttp ( 100 mm ) , 2 l of taq polymerase ( 5 u/l ) . it was mixed by gentle vortex , centrifuged shortly and incubated at 72 c for 2 h. t - vectors were separated from the self - ligated and concatemerized plasmid dna on a 1% agarose gel by 0.5 g / ml of ethidium bromide . the dna bands were visualized by a hand - held long wave ( 365 nm ) uv light . the gel slice transferred to a 2 ml micro centrifuge tube , and then t - vectors were purified and recovered by silica bead dna gel extraction # k0513 ( fermentas co. ) via the kit manufacturer s guidelines . the density of the purified t - vectors was determined by running 1 l of the t - vector in compared to standard dna ladder in a 1% agarose gel at 5 volts / cm for 45 min and then compared the comparative brilliantness of the bands . . 1:3 molar ratio of t - vector to insert dna was recommended , but the amount of a - tailed dna solution in a 10 l ligation should not be more than 2 l . in a sterile 0.5 ml micro centrifuge tube , added : 2 l t - vector , 2 l of a - tailed dna , 1 l of 10 x ligation buffer , 1 l of t4 dna ligase ( 23 u/l ) and 4 l of ddh2o . it was mixed gently , centrifuged briefly and incubated at 14 c for overnight ( fig . before transformation , luria - bertani ( lb ) agar plates contained 100 g / ml of ampicillin , were prepared and spreaded with 5-bromo-4-chloro-3-indolyl--d - galactoside ( x - gal ) and isopropylthio--d - galactoside ( iptg ) . for each ligation reaction , 50 l aliquoted of frozen competent cells ( top 10 ) was thawed on ice and added 2 l of the ligation reaction to the competent cells and mixed gently by stirring with the pipette tip . the cells were incubated on ice for 30 min then heats shocked for 30 s in the 42 c water bath , and then were immediately placed on ice for 2 min . the transformed cells were spreaded on each labeled lb - ampicillin plate with x - gal and iptg . the positive colonies that had white color were identified either by restriction enzyme and screening by pcr method after transformation . pcr screening was carried out with same primers that be used for pcr amplification for insert production . for screening by restriction enzyme method , the insert was released by digestion with two unique restriction enzymes ( with cutting sites , ecor1 : gaattc and bamh1 : ggatcc ) from the multiple cloning sites , and the insert size was confirmed by agarose gel electrophoresis ( fig . pcr product of pvmsp-1 42 kda was directly analyzed on abi prismtm 310 automated sequencer to determine p. vivax variation ( strain / haplotype ) in the infected patient . both directions of our sequences were aligned and edited using sequencher tm 4.0.4 software for pc ( gene codes corporation ) and mega 5 software for phylogenetic analysis and then were compared with some genbank sequences for their similarity ( 10 ) . a phylogenetic hypothesis of p. vivax was generated with pvmsp-1 42 kda using the nonparametric bootstrapping , neighbor joining method . in fig . 1 , intact plasmid ptz57r , digested plasmid ptz57r by ecorv and digested recombinant ptz57r/ msp by bamhi and eco - ri restriction enzymes and expected insert band are presented . as fig . 2 shows an 1124 bp band has been demonstrated from extracted genomic dna by pcr . lane 1 : pv msp-142 kda gene was nearly 1124bp long , lane 2 : size marker 1 kb the yield of pcr was cloned in ptz57r and then sequenced . in the iranian p. vivax isolate , as compared to the reference sal i sequence , 38 snp of pvmsp-142 
 kda , were identified . as fig . 4 shows , msp-1 42 kda phylogenetic tree based on bootstrap - neighbor joining method in iranian sequenced isolate ( msp 42-yyxxxxxx - iran 01 ) and other recorded sequences in genbank . all of the mutations were localized in the pvmsp-133kda , while there was almost no variation in the msp-119 kda . 2 out of 38 mutations ( haplotype ) were novel in the 545 and 573 positions as compared to the homogeneous sequences from other surveyed countries ( fig . 3 , 4 ) . bootstrap neighbor - joining consensus tree of pv - msp-142 kda data set by mega5 . sequence of iranian isolate is shown for msp 42-yyxxxxxx - iran 01 in clad of indian and japonica sequences ( accession nos : eu 430479.1 , af435635.1 ) sequence alignment of pv msp-142 kda gene in p. vivax isolated from iran and correlated part of the isolated genes from other countries in research on diagnostic potential for p. vivax malaria parasite subunits , the focus is on the c - terminal region of p. vivax msp-1 . as fig . 5 depicts , generally , msp1 is manufactured as a big protein ( 200 kda ) which is connected to the cell membrane by a glycophosphatidylinositol ( gpi ) anchor in the carboxyl terminus region ( 11 ) . msp1 undergoes a series of proteolytic maturation changes at the same time as the merozoite unleashed from ( rbc ) and generates four polypeptide fragments of nearly 83 , 30 , 38 , and 42 kda from the n - terminus to c - terminus ( 12 ) , which stays as interconnected parts on the cell surface by the anchored c - terminal segment ( p42 ) ( 13 ) . new rbcs are invaded rapidly by attachment of free merozoites induces second in a series cleavage of the p42 peptide to generate p33 , which is shed together with the previous fragments and p19 , abides by anchoring to the membrane of parasite as it invades the cell ( 14 , 15 ) . high plentifulness and important function on the cell surface , probably caused msp1 to be a principal object of the host immune system and antibodies are identified on different regions of this protein ( 16 ) . antibodies that recognize the c - terminal region of plasmodium falciparum msp-1 , inhibit the invasion of merozoites into the host erythrocytes in vitro and immunization of experimental animals with msp-119 
 kda also confers protective immunity ( 17 , 18 ) . these findings demonstrate that msp-142 
 kda is a promising candidate antigen for blood stage vaccine development and diagnostic kits . panel ( a ) shows primary processing , and ( b ) shows secondary processing in this study , the pv msp-142 
 kda was cloned , sequenced , and subsequently compared with homogeneous genes that previously had been recorded in genbank . 38 snp of pvmsp-142 kda , were recognized using the sequencher 4.0.4 and mega 5 programs . pacheco et al . in indonesia tried to show the genetic diversity of the 42 kda fragment of msp-1 antigen in p. vivax . they found that the msp-133 
 kda fragment exhibits greater genetic diversity than the msp-119 kda generally . previous observations confirmed that the msp-119 kda fragment is more conserved than the msp-133 kda fragment ( 19 ) . in present study , polymorphism of p. vivax msp-133 kda is not evenly spreaded , indeed there is a region of 70 bp in msp-133 kda where a clear excess of non - synonymous is observed in the overall msp-133 
 kda results , while there is closely no significant variation in the msp-119 
 kda . two out of 38 mutations were new haplotypes ( 545 and 573 positions ) in the 33 kda fragment as compared to the homogeneous sequences from other countries including japan , turkey , usa , india , sumatra , vietnam , indonesia , riometa , china , france and reference sequence from sal - i ( 20 ) ( fig . 3 ) . due to the differentiation of msp1 amino acid sequences between plasmodium spp . , it is necessary to use recombinant msp-1 to detection antibody from p. vivax in iranian cases ( 21 ) . compared the p. vivax msp-3 gene with ssrrna gene as genetic markers for the parasite detection . according to their results the sensitivity of ssrrna gene was higher than the pvmsp-3 gene ( 100% vs. 95% ) . they concluded that the pvmsp-3 gene can not be a suitable marker for detection of p. vivax in blood sample by pcr ( 22 ) . one of the prominent problems in diagnostic kits is genetic polymorphisms that encoding on this region , within and between the p. vivax population . therefore , polymorphism examination of this gene on disparate territories could be helpful in improving diagnostic kits based on antibody detection in malarious areas of iran . shahbazi and colleagues in iran assessed the genetic structure of p. vivax population by sequence analysis of the merozoite surface protein 3 ( msp-3 ) gene . moreover , their phylogenetic analysis did not show any significant geographical branching ( 23 ) . studied the p. vivax dihydrofolate reductase ( pvdhfr ) mutations among 50 blood samples of symptomatic patients from 4 separated geographical regions of south - east iran . they reported point mutations at residues 57 , 58 , 61 , and 117 by using the pcr - rflp method . polymorphism at positions 58r , 117n , and 117 t of pvdhfr gene has been found in 12% , 34% , and 2% of isolates , respectively . one of the endemic zones for p. vivax throughout the world is iran from which there are reported cases annually ( 25 ) . asymptomatic carriers of p. vivax among treated patients have been shown as a major reservoir of parasites and maintenance of high levels of transmission in malarious areas of iran ( 26 ) . although genetic polymorphisms in the regions of msp-1 in iranian p. vivax isolates has been previously analyzed , but little information is available regarding the genetic polymorphism of msp-142 
 kda among iranian p. vivax isolates . in this study , the genetic polymorphism of msp-142 
 kda in an iranian isolate was analyzed for a better understanding of the nature of iranian p. vivax . thus , it is significant to investigate the molecular character of the parasite placed within iran and comparing with the same species in other regions . predicted protein sequence of cloned pvmsp-142 
 kda gene obviously has a high degree of identity ( 97% ) with strong homology to the pv msp-142 
 kda gene of p. vivax . asian isolates specially japan and india also have sequence similarity ( 95% ) pv msp-142kda gene from sal - i isolate ( as reference sequence ) . in general , diversity of anopheles mosquitoes , presence of several plasmodium species , biotic interactions and hybrid / mixed infections can affect the genotyping variation of plasmodium in low to high degree . this might result in parasite pathogenicity , host specificity , establish and persist of infection , different clinical manifestation , transmission dynamics and antigenicity of infection . the cloned pvmsp-142 
 kda gene can be used as a strong candidate for diagnostic kits based on antibody , antigen detection and vaccine development .