VIR-C2 is a member of subfamily C. 2]. Endemic regions in the Republic of Korea (ROK) are mainly found around the demilitarized zone at the border with the Democratic Peoples Republic of Korea. The area had been declared malaria-free by ROK and the World Health Business in the 1970s, but malaria cases re-emerged in the 1990s [3]. While efforts have been taken to treat and prevent this disease, more than 500 cases occur every year up to this point. Moreover, the introduction of chloroquine-resistant has been reported in ROK [4]. An effective malaria vaccine capable of inducing a strong and long-lasting protection in naturally uncovered individuals should be sought after. Studies evaluating immune responses against different antigens will aid in the process of vaccine development. Variant surface antigens (VSAs) of many spp. are the key proteins used by the parasites to escape from the host immune system [5]. erythrocyte membrane protein 1 (PfEMP1) and variant interspersed repeat (VIR) proteins encoded by multigene families located on telomeric and subtelomeric regions of the parasites chromosomes have been acknowledged in and genes are divided into 12 subfamilies, named A to L [7, 8]. VIR proteins are exported to the infected erythrocytes surface for host immune system evasion, 6-Carboxyfluorescein and in reticulocytes, they partially induce the infected cells adherence to the endothelial cell receptors [9C11]. VIR antigens are also reported to induce the natural acquisition of antibody-producing and T cell memory responses to parasites important in uncovered and pregnant populations [12, 13]. In ROK, previously, four VIR genes were studied to understand their genetic diversity, in which the genes showed moderate diversity levels [14]. However, immune responses induced by 6-Carboxyfluorescein VIR proteins in is usually endemic in the summer season (June to August). The admission and clinical management of the patients were undertaken independently after blood collection and diagnosis. A total of 681 venous blood samples were collected between 2011 to Rabbit polyclonal to APBA1 2019. In some samples, the parasite was not observed using microscopy due to low parasitaemia, even though the rapid diagnostic test and nested-polymerase chain reaction (PCR) were positive [15]. The blood samples were centrifuged at 1500for 15?min to separate erythrocytes and serum for 6-Carboxyfluorescein further studies. Among the 681 patients, more than 15?ml of venous blood samples were collected aseptically in heparinized tubes from 25 patients (10, 10, and 5 samples from 2017, 2018, and 2019, respectively) for the proliferation and cellular activation assay. Samples were also obtained 6-Carboxyfluorescein from 30 healthy volunteers who resided in non-endemic areas (southern 6-Carboxyfluorescein parts) of the ROK and had not travelled to endemic areas. The plasma samples were transported on ice to a laboratory in the Department of Parasitology and Tropical Medicine at Kyungpook National University and were stored at ??70?C until use. Recombinant VIR proteins, MSP1-19 protein and two synthetic peptides The seven VIR (VIR-A4, VIR-B10, VIR-C1, VIR-C2, VIR25, VIR14 and VIR2) proteins and two synthetic peptides previously used to probe for an immune response against were prepared for this study [12, 13]. Recombinant VIR proteins were generated as glutathione S-transferase fusion proteins per protocol described previously with modifications [5]. Briefly, PCR products were inserted in the pGEX-4T-3 vector (GE Healthcare, UK), and the sequences were confirmed by standard double-stranded DNA sequencing before expression. Recombinant BL21 (DE3) was produced at 37?C and 250?rpm in multiple flasks containing 500?ml of LB-ampicillin. When the preparation reached an OD600?=?0.4C0.6, isopropyl–d thiogalactopyranoside (IPTG, Invitrogen, New Zealand) was added to a final concentration of 2?mM. Cultures were incubated at 18?C and 250?rpm for 16?h, and bacterial pellets were obtained by centrifugation and resuspended in appropriate volume of sonication buffer [10?mM TrisCHCl pH 8.0, 150?mM NaCl, 1?mM EDTA, 100?g/ml lysozyme, and 1% Triton X-100]. Bacteria were lysed on ice with the aid of.