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. 2020 Apr 20;10:6573. doi: 10.1038/s41598-020-63611-6

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Assessment of truncated recombinant PfRipr for GIA. (a) Design of PfRipr truncates. Eleven overlapping fragments of PfRipr were designed as shown. (b) Expression of recombinant PfRipr truncates. PfRipr truncates were expressed by WGCFS as GST-fused proteins with a C-terminal His-tag, Ni²⁺ affinity purified, resolved by 12.5% SDS-PAGE under reducing conditions, and stained with CBB. Lanes 1–11 represent respective PfRipr truncates shown in Fig. 2a. M: molecular weight marker. (c) GIA activity of antibodies against PfRipr regions. The GIA activity of 10 mg/ml rat antibodies to each of the 11 PfRipr regions on P. falciparum 3D7 strain was compared. His-GST: negative control, anti-His-GST antibodies; PfRipr: anti-Ecto-PfRipr antibodies; 1–11: antibodies against each PfRipr truncate shown in Fig. 2b. Results are mean ± SEM of pooled data from six independent experiments. Error bar represents standard error of the mean. * Indicates statistically significant (Kruskal-Wallis test; P < 0.05) when compared against antibodies to His-GST. (d) GIA with purified PfRipr_5 antibodies and anti-PfRipr antibodies-depleted flow-through fraction. Eluate (0.72 mg/ml) and flow-through (10 mg/ml) fractions of rabbit anti-PfRipr K₂₇₉-D₉₉₅ antibodies purified with a recombinant PfRipr_5 immobilized column were compared in GIA. His-GST: rabbit anti-His-GST antibody as negative control; PfRipr K₂₇₉-D₉₉₅: anti-PfRipr K₂₇₉-D₉₉₅ antibodies, each at 10 mg/ml. Results are mean ± SEM of pooled data from three independent experiments. Error bar represents standard error of the mean. * Indicates statistically significant (Kruskal-Wallis test; P < 0.05) when compared against antibodies to His-GST. (e) Expression of PfRipr_5 truncate protein using baculovirus protein expression system (PfRipr_5-BPES). Purified recombinant PfRipr_5-BPES was resolved by SDS-PAGE under reducing conditions and stained with Coomassie brilliant blue (CBB). M: All Blue prestained protein molecular weight marker. (f) Reactivity of anti-PfRipr_5-BPES antibody on parasite PfRipr. Western blot analysis of PfRipr in trophozoite and schizont-rich parasite lysate using rabbit anti-PfRipr_5-BPES antibodies. M: molecular weight marker. (g) Dose dependent GIA activity of rabbit antibodies to PfRipr_5. GIA activities of anti-PfRipr_5 antibodies generated by BPES-expressed recombinant protein were measured at two-fold dilution; range 20 to 0.31 mg/ml. Solid red line, antibodies against PfRipr_5-BPES immunized at 0.1 mg/dose; dashed red line, antibodies against PfRipr_5-BPES immunized at 0.3 mg/dose.