| The apical membrane antigen-1(AMA-1) and Major Merozoite Surface Protein-1 C-terminal region (19kDa) of Plasmodium falciparum(P. falciparum) are the most important asexual blood stage antigens. Among the blood-stage antigens identified ,apical membrane antigen 1(AMA-1)has emerged as one of the leading target vaccine candidates.The AMA-1 of P.falciparum is an 83-kDa polypeptide with characteristics of an integral membrane protein.AMA-1 contains a hydrophobic signal sequence of 23 amino acids in the C-terminal region that correspond to a transmembrane domain and has a 55 amino-acid cytoplasmic tail. Ectodomain of AMA-1 contains three integrated disulfide-bonds domain, consist of sixteen conserved cysteines, The third domain AMA-1(III) is high conserved in sequences. It was estimated that AMA-1(III) domain was still carried by merozoite, probably involved in the invasion of merozoite. Thus, AMA-1(III) is regarded as one of the most important asexual blood stage vaccine candidates against malaria infection. The major surface protein 1(MSP-1) has also been implicated as a target for protective immunity by a variaty of criteria in studies of P.falciparum in nonhuman primates and in vitro studies of laboratory parasite host models, and aeroepidemiological studies of naturally acquired immunity. In several studies, MSP-1 purified from P.falciparum partially or completely protected against challenges in primate models. Partial protection was also observed with peptides or recombinant proteins. Monoclonal antibodies to MSP1 inhibit parasite development in Vitro. The C terminus of MSP1, from which MSP1-19 is derived, has been studied in most detail as the target of a protective immune response. The available evidence suggests that antibody against MSP1-19 can inhibit parasite growth in vivo and in vitro. Thus, the major Merozoite Surface Protein 1- MSP1, especially MSP1-19 of P.falciparum is also regarded as a leading vaccine candidate for malaria. Objectives Immunization and vaccine trials require not only large amounts of the protein, but also highly purified homogenous protein as well. In order to obtain enough high quality purified AMA-1(III) and MSP1-19 proteins in this study, we have expressed the AMA-1(III) and MSP1-19 genes in yeast system of pichia pastoris at competitive level and have produced the AMA-1(III) and MSP1-19 recombinant Proteins. Materials and methods The experiments were composed of two parts. 1. Expression and Purification of Apical Membrane Antigen-1 of P. falciparum in yeast pichia pastoris. 2. Expression and Purification of Major Merozoite Surface Protein-1 C-terminal region (19kDa) of P. falciparum in yeast pichia pastoris. Firstly, the AMA-1(III) and MSP1-19 genes were amplified by PCR technique from recombinent vector pPF4/PFCP2 and were inserted into cloning vector pPF4 and pPIC9, respectively. The validity of these sequences was confirmed by automatic DNA sequencing. After then, recombinant expression vectors were constructed by ligating them with pPIC9K vector. By using electroporation transformation methods, recombinant expression vectors containing AMA-1(III) and MSP1-19 genes were transformation into pichia GS115 cells, His+Muts transformants were selected with RDB plates and the multiple inserts were screened using different concentrations of G418. Proteins expression of AMA-1(III) and MSP1-19 were analysised by SDS-PAGE and Western blot as well. The integration of AMA-1(III) and MSP1-19 genes in the transformant's genome was assessed by genomic PCR. After big batch culturing using 1000ml baffled flask,the concentrated supernatant was purified by Ni-NTA chromatogragh method. The expression form and relative molecular weight of AMA-1(III) and MSP1-19 proteins were identified by reducing SDS-PAGE. Results First of all, P. falciparum AMA-1(III) and MSP1-19 genes were successfully cloned by PCR and recombinant vectors pPF4/AMA-1(III),pPIC9/AMA-1(III),and pPF4/MSP1-19, pPIC9/MSP1-19 were constructed by inserting these genes into pPF4 and pPIC9, respectively...
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