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PHD : Parasites, Hosts and Diseases

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Original Article

Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening

The Korean Journal of Parasitology 2015;53(4):403-411.
Published online: August 25, 2015

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Corresponding author (ethan@kangwon.ac.kr)
• Received: February 28, 2015   • Revised: July 23, 2015   • Accepted: July 23, 2015

© 2015, Korean Society for Parasitology and Tropical Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening
Korean J Parasitol. 2015;53(4):403-411.   Published online August 25, 2015
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Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening
Korean J Parasitol. 2015;53(4):403-411.   Published online August 25, 2015
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Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening
Image Image
Fig. 1. Schematic protein features and secondary structures of the PvRBP family. The PvRBP family based on amino acid (aa) length. Black bar denotes fragment for recombinant protein expression, with the order of fragment number from the N-terminal region. RDG, arginyl-glycyl-aspartic acid a cell adhesion motif.
Fig. 2. Recombinant protein expressions of PvRBP family fragments by Western blot analysis. All the PvRBP recombinant proteins were expressed in the supernatant as the soluble fraction with variable expression level in the wheat germ cell-free expression system. The expression level of each PvRBP protein was detected by Western blot analysis probed with anti-His tag. kDa, kilodalton; T, total crude protein; S, supernatant soluble fraction.
Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening
Name Gene ID (PVX_) Chromosome No. Length (aa) MW (kDa) SP TM Expression stage Note Reference
PvRBP1a 098585 7 2,833 326.2 Y Y Schizont Full length [19,23]
PvRBP1b 098582 7 2,608 303.3 Y N Unknown Full length [14]
PvRBP1 like-partial 125738 NA 785 90.9 N Y Unknown Partial length [14]
PvRBP2a 121920 14 2,487 286.6 Y N Schizont Full length [14,21]
PvRBP2b 094255 8 2,806 325.5 Y N Schizont Full length [14,21]
PvRBP2c 090325 5 2,824 326.4 Y Y Schizont Full length [19,23]
PvRBP2 like-partial A 090330 5 623 72.8 Y N Schizont Partial length [14,21]
PvRBP2 like-partial B 101590 14 640 74.2 Y N Unknown Partial length [14,21]
PvRBP2 like-homologue B 116930 12 1,060 123.9 Y N Unknown Full length [14,21]
PvRBP2d 101585 14 2,829 329.1 Y Y Null Pseudogene [21]
PvRBP3 101495 14 2,798 323.1 N Y Null Pseudogene [21]
Fragment (PVX_) Forward primer (5ʹ→3ʹ) Reverse primer (5ʹ→3ʹ) Position (aa) Length (aa) pI MW (kDa)
098585-1 CAAAACAGCATGCAGCAGTAC TTCGTCTACTTCCCTTTTGATGT 804-1,073 270 5.1 34.4
098585-2 GAAATGAATTCTAAAAAGAGCGCT TTCTTCTTTGTTTTCTGGGTTCA 2,619-2,822 204 4.2 25.6
098582-1 GTAGATTTAAATCGAGGGAAAAACAC GCAGCTGGATTGCTTCAAA 15-289 273 6.7 34.7
098582-2 GATAAGTTGAAAGCAGAACTAGGAAGT GATCTGTTGTGTTTGCTCTTCC 996-1,254 259 5.6 33.8
125738-1 CTAGAAAATGACAAAAGGAAAAAGAGC AATGTCTGTTTGAGGGGGTAGTC 476-623 148 4.6 20.0
125738-2 AAAAATGACAGAAACGATCAAAAAT ATCTTTTACATTACGTTTTGAACCATT 630-772 143 5.4 18.9
121920-1 TCAAGCAAAGAAAGCAATCG TATTTGATTCTTTGGTGTGAAGAGAA 20-145 126 9.7 17.7
121920-2 AGCGAAAAAATAGATAAAAATTTGGA CATATCGTCGACTAATTCTTTTAAGTC 453-713 261 5.2 33.3
121920-3 ACGGAAGCTAAAACACTCAGG TGTGTCGTAATTCTTATCATCCGT 1,789-2,049 261 5.8 32.9
121920-4 GCTAAGGAAAACTCCATTAATATGC TTCTTCCTTATCTTGAAGATTTCCA 2,330-2,474 145 7.4 19.0
094255-1 CAAGAAGCTTCCTACCAAAATGC TGCAATATTCACTTTCTTAATGGAGT 728-965 238 4.7 30.7
094255-2 GATACCCAAGAGAACGATACAGAC TTCAGAATAATCGCTATTGTCGAA 2,673-2,806 134 4 18.2
090325-1 AGGAATGGTCAACACAAATACAAC TACAATTTCAGGGGCCTCTG 47-171 125 9.7 18.0
090325-2 CTAAAAGCAAGCTCGAACGATC GTATTTATCAGCATTTGATTTCATTTC 2,115-2,314 200 5 25.7
090325-3 GCAAAAGTTGAGCCTGAAGC ATCTTCTTCATTAAAGCAAACTTCG 2,652-2,824 173 3.9 22.3
090330-1 AGGGATGATCAAAACGGACA TGAATTTGGAACACTATGCAATTC 47-145 99 8.4 14.4
090330-2 GAAAATCCAGAACACTATAAAAATAAGAGA AATTTCATCTATTACGAAATCTGCC 242-370 129 6.8 18.5
101590-1 AAGGATGTCAATCGAAACAAACC ATGCAATTTTTTATTATCATCTTCCAT 22-145 124 7.5 17.2
116930-1 TTGCACAATTCGACCTCC AACCATGCTGCCTTCGTAC 359-477 119 6.3 16.6
116930-2 GATAACATCAAGAGGAGGAACGC CAGTTTGAAATTTTTATCGATGTTGT 603-736 134 9.7 19.4
Antigen (PVX_) No. of patients samples (n)
95% Cib (%) MFIc No. of healthy samples (n)
95% CI (%) MFI P-valuee Features/Structures
Posit. Neg. Total (%)a Posit. Neg. Total (%)d
098585-1 10 6 16 (62.5) 38.6-81.5 6,429 0 8 8 (100.0) 67.6-100.0 3,139 0.0013 Repeat sequence
098585-2 8 8 16 (50.0) 28.0-72.0 5,480 0 8 8 (100.0) 67.6-100.0 2,943 0.0016 C-terminal
098582-1 6 10 16 (37.5) 18.5-61.4 5,806 0 8 8 (100.0) 67.6-100.0 3,275 0.0011 N-terminal
098582-2 10 6 16 (62.5) 38.6-81.5 6,566 0 8 8 (100.0) 67.6-100.0 3,181 0.0004 Coiled coil
125738-1 11 5 16 (68.8) 44.4-85.8 9,242 0 8 8 (100.0) 67.6-100.0 3,970 0.0002 Coiled coil
125738-2 11 5 16 (68.8) 44.4-85.8 10,280 0 8 8 (100.0) 67.6-100.0 3,741 0.0003 C-terminal
121920-1 11 5 16 (68.8) 44.4-85.8 11,283 0 8 8 (100.0) 67.6-100.0 4,628 0.0013 N-terminal
121920-2 11 5 16 (68.8) 44.4-85.8 9,232 0 8 8 (100.0) 67.6-100.0 3,863 0.0007 Low complexity
121920-3 11 5 16 (68.8) 44.4-85.8 8,821 0 8 8 (100.0) 67.6-100.0 3,010 0.0005 Repeat sequence
121920-4 6 10 16 (37.5) 18.5-61.4 6,947 0 8 8 (100.0) 67.6-100.0 3,710 0.0053 C-terminal
094255-1 12 4 16 (75.0) 50.5-89.8 13,325 0 8 8 (100.0) 67.6-100.0 5,163 0.0007 Repeat sequence
094255-2 8 8 16 (62.5) 28.0-72.0 6,307 0 8 8 (100.0) 67.6-100.0 3,394 0.0011 C-terminal
090325-1 14 2 16 (87.5) 64.0-96.5 17,597 0 8 8 (100.0) 67.6-100.0 6,257 0.0002 N-terminal
090325-2 9 7 16 (56.3) 33.2-76.9 19,179 0 8 8 (100.0) 67.6-100.0 7,226 0.0004 Short coiled coil
090325-3 11 5 16 (68.8) 44.4-85.8 18,615 0 8 8 (100.0) 67.6-100.0 6,095 0.0007 C-terminal
090330-1 13 3 16 (81.3) 57.0-93.0 12,413 0 8 8 (100.0) 67.6-100.0 4,455 0.0002 N-terminal
090330-2 7 9 16 (43.8) 23.1-66.8 12,149 0 8 8 (100.0) 67.6-100.0 6,072 0.0030 Short coiled coil
101590-1 9 7 16 (56.3) 33.1-76.9 7,223 0 8 8 (100.0) 67.6-100.0 3,299 0.0005 N-terminal
116930-1 12 4 16 (75.0) 50.5-89.8 12,407 0 8 8 (100.0) 67.6-100.0 4,522 0.0004 Short coiled coil
116930-2 14 2 16 (87.5) 64.0-96.5 11,332 0 8 8 (100.0) 67.6-100.0 3,346 0.0005 Repeat sequence
PvMSP1-19 4 0 4 (100.0) 51.0-100.0 9,987 0 8 8 (100.0) 67.6-100.0 1,887 0.0286
Table 1. General information of Plasmodium vivax reticulocyte binding like protein (PvRBP)

aa, amino acid; MW, molecular weight; kDa, kilodalton; SP, signal peptide; TM, transmembrane domain; NA, not assigned.

Table 2. Primer sequences for recombinant PvRBP family protein expressions

pI, isoelectric point.

Table 3. Total IgG prevalence of each recombinant PvRBP protein including predicted B-cell epitope domain

Sensitivity, percentage of positives in patient samples.

CI, confidence intervals.

MFI, mean fluorescence intensity.

Specificity, percentage of negatives in healthy samples.

Differences in the total IgG prevalence for each antigen between patients and healthy individuals were calculated with Mann-Whitney U test. P<0.05 considered as statistically significant.