en Original article Original article <p> <strong> Background: </strong>The purity and correct folding of a recombinant protein is critical for any structural, biochemical and vaccine design studies. <i>Plasmodium vivax</i> Duffy binding protein-II is a leading vaccine candidate for vivax malaria. In the present study, the purification process of recombinant DBP-IX (a variant form of PvDBP-II) was optimized to achieve the highest yield and purity. Moreover, naturally-acquired IgG antibodies to the expressed protein have been evaluated. <strong>Material and Methods: </strong>DBP-IX was cloned and expressed as a his-tagged protein in <i>E. coli</i>. The recombinant protein was purified using Ni-NTA agarose and different purification parameters were optimized to achieve the highest yield and purity. The quality of the purified rDBP-IX was assessed by different procedures such as SDS-PAGE gel analysis in both reducing and non-reducing conditions, followed by indirect immunofluorescence antibody test and ELISA using the sera of <i>P. vivax</i> infected patients (n= 202).<strong> R esults: </strong>DBP-IX was successfully cloned, expressed and optimally purified. Differential mobility of the rDBP-IX on the SDS-PAGE gel in reducing and non-reducing conditions, confirmed the presence of disulphide bonds. In addition, anti-rPvDBP-IX antibody produced in mice recognized the native PvDBP-II, suggesting that epitopes in the recombinant protein were similar to the corresponding native form. Finally by performing ELISA experiments, it was demonstrated that natural <i>P. vivax</i> infection produces IgG against rDBP-IX (42.1%) whilecytophilicIgG1 antibody (35.4%) was the predominantly detected IgG subclass.<strong> Conclusion: </strong>The results indicated that the optimally-purified rDBP-IX was of a quality that could be used in vaccine development research and immunological studies of vivax malaria. </p> Plasmodium vivax, Duffy binding protein, Recombinant protein, Vaccine 1 6 http://vacres.pasteur.ac.ir/browse.php?a_code=A-10-27-10&slc_lang=en&sid=1 Vahideh Valizadeh No Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran Sedigheh Mirkazemi No Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran Behrouz Vaziri No Department of Medical Biotechnology, Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran Sedigheh Zakeri zakeris@pasteur.ac.ir, zakerimvrg@gmail.com Yes Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran Navid D. Djadid No Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran