• Parasites, Hosts and Diseases
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• v.54 no.4
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• pp.385-391
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• 2016

The discovery and understanding of antigenic proteins are essential for development of a vaccine against malaria. In Plasmodium falciparum, Pf92 have been characterized as a merozoite surface protein, and this protein is expressed at the late schizont stage, but no study of Pv92, the orthologue of Pf92 in P. vivax, has been reported. Thus, the protein structure of Pv92 was analyzed, and the gene sequence was aligned with that of other Plasmodium spp. using bioinformatics tools. The recombinant Pv92 protein was expressed and purified using bacterial expression system and used for immunization of mice to gain the polyclonal antibody and for evaluation of antigenicity by protein array. Also, the antibody against Pv92 was used for subcellular analysis by immunofluorescence assay. The Pv92 protein has a signal peptide and a sexual stage s48/45 domain, and the cysteine residues at the N-terminal of Pv92 were completely conserved. The N-terminal of Pv92 was successfully expressed as soluble form using a bacterial expression system. The antibody raised against Pv92 recognized the parasites and completely merged with PvMSP1-19, indicating that Pv92 was localized on the merozoite surface. Evaluation of the human humoral immune response to Pv92 indicated moderate antigenicity, with 65% sensitivity and 95% specificity by protein array. Taken together, the merozoite surface localization and antigenicity of Pv92 implicate that it might be involved in attachment and invasion of a merozoite to a new host cell or immune evasion during invasion process.

• Parasites, Hosts and Diseases
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• v.53 no.4
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• pp.403-411
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• 2015

Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (>326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.

• Korean Journal of Veterinary Service
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• v.25 no.3
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• pp.245-257
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• 2002

The gene encoding the HN protein from the CBP-1 strain, a heat stable Newcastle disease virus (NDV) isolated from diseased pheasants in Korea, was characterized by reverse transcriptase- polymerase chain reaction(RT-PCR) and the nucleotide and amino acid sequences were analyzed following cloning of the HN gene. In all of the NDV strains studied, a 1.75 kb size cDNA fragment for the HN gene was generated by RT-PCR and smaller specific band sizes harboring the internal portions of the HN gene were also detected by using four pairs of primers. The RT-PCR was sensitive enough to detect viral transcripts when the virus titer was above 25 hemagglutination units. The amplified 1.75 kb cDNA was cloned into a BamHI site of the pVL1393 Baculo transfer vector. The nucleotide sequences of the 1,758 bp HN gene from the CBP-1 strain were determined by the dye terminator cyclic sequencing method. The gene sequences were compared among the strains of CBP-1, Texas GB, Beaudette C, LaSota, B1 and Ulster. The homology of the CBP-1 HN gene to other HN variants was 97.8% to Texas GB, 98.4% to Beaudette C, 95.4% to LaSota, 95.6% to B1 and 90.2% to Ulster. As the deduced 577 amino acid sequences were compared among the strains, the homology for CBP-1 HN appeared to be 96.7% to Texas GB, 97.9% to Beaudette C, 95.5% to LaSota, 95.5% to B1 and 92.7% to Ulster. It was evident that the amino acid sequences included 5 sites for N-asparagine linked glycosylation and 12 cysteine residues. The three conserved leucine residues within the predicted transmembrane domain of the HN protein are amino acid 30, 37 and 44. The three antigenic sites on the HN protein of NDV are amino acids 347(Glu), 481(Asn) and 495(Glu). These data indicate that the genotype of the CBP-1 strain is more closely associated with the strains of Texas GB and Beaudette C than it is for the LaSota, B1 and Ulster strains.