Background is responsible for approximately 80 million malaria cases in the world. amplified by PCR and cloned in pUC19 vector for sequencing. Results Sequence analysis of 859-18-7 manufacture the antigen showed a high degree of identity (99%) with strong homology to the PvAMA-1 gene of and isolates from India and Korea (Asian isolates) respectively, and 96% similarity with AMA-1 gene from El Salvador. Conclusions We cloned and characterized three domains of PvAMA-1 gene from an Iranian patient. Predicted protein sequence of this gene showed some discrepancies in corresponding protein in comparing with similar genes reported from other malarious countries. and about 2.6 billion people are at risk of the infection in the world (1). The infection causes approximately 132-391 million episodes of disease each year (2). Although is the main responsible for malaria morbidity outside Sub-Saharan Africa, it has received little attention and limited funds for research and control owing to producing less severity than (3). Moreover, non-cultivable nature of has induced some limitations for the study of its molecular biology up to now. Indeed, increasing the knowledge of genetic diversity and population structure of this parasite will result in the development of an effective vaccine to control the disease (4). Apical membrane antigen (AMA-1) is a member of a group of molecules which are quite conserved in species (5) and are expressed on the micronemes and surface of merozoite in all species (6). AMA-1 is expressed in the late schizont stage of the asexual plasmodia life cycle (7). The antigen plays a unique role in NGF critical stage of invasion the human hepatocytes by sporozoites (8) and red blood cells by merozoites (9C12). This gene shows few predominant haplotype and displays very limited genetic diversity within any geographic region so more investigations are needed to confirm these observations (13). Moreover, AMA-1 is one of the most immunodominant antigens which are considered as promising antigen for the development of a recombinant vaccine against (7, 14, 15). It could be also used for immunological studies to evaluate the natural acquired antibody response to 859-18-7 manufacture AMA-1 (16). Therefore, polymorphism investigation of this gene in different areas could result in developing diagnostic kits. Iran is one of the endemic areas for infection. Blood of the patient was collected into the EDTA tube using venipuncture method. infection was documented by microscopic analysis using Giemsa stained thick and thin blood smears. DNA extraction The genomic DNA was extracted from the whole blood using Genomic DNA Extraction Kit, ACCUPreP? kit (BIONEER) according to the manufacturer’s instructions. The quality and quantity of the extracted DNA was checked using a biophotometer (Ependorf) at 260 and 280 nm and electrophoresis on 1% agarose gel. PCR amplification of PvAMA-1 gene A partial region of PvAMA-1 gene, including domain I, domain II and domain III, covering amino acids (AA) residue 42C488 was targeted for amplification. The primers were designed based on the sequence of apical merozoite antigen- 1 (PvAMA-1gene, ACCESSION NO.: “type”:”entrez-nucleotide”,”attrs”:”text”:”XM_001615397″,”term_id”:”156098862″,”term_text”:”XM_001615397″XM_001615397) with the following sequences: PvAMAF (5-CCATGGGGCCTACCGTTGAGAGAA-3) and PvAMAR as (5- CTCGAGTCATAGTAGCATCTGCTTGTT- 3). The PCR was performed in a 200 l thin-walled PCR tube containing: 200 ng extracted DNA as template, 20 pmol of each primer, 12.5 l of Pwo master PCR master mix (Roche) and ddH2O up to 25l. The targeted gene was amplified for 30 cycles (95C for 20s, 58C for 30s and 72for 2 min) and the PCR product was checked on 1.0% agarose gel against DNA size marker (Fermentase), then 859-18-7 manufacture it was purified from agarose gel by Qiaquick gel extraction kit (Qiagen) to be prepared for cloning. Cloning and sequencing of the PvAMA-1 gene Purified DNA fragment was inserted into a SmaI cut pUC19 plasmid using rapid DNA ligation kit (Roche) according to the manufacturer’s protocol. The transformation was done in CaCl2 treated competent cells of Novablue using thermal shock method (30min at 0C, 2min at 42C and 2min at 0C). The transformants were incubated at 37C for 45 min and cultured on the LB agar plates containing 100 g/ml ampicillin, 1 mM IPTG and 50 g/ml X-gal and incubated for.