All authors approved the manuscript. Data availability The datasets generated during and/or analyses during the current study are available from the corresponding author upon request. Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Contributor Information Hiroshi Tachibana, Email: pj.ca.iakot-u.cci.si@abihcath. Somchai Jongwutiwes, Email: moc.liamg@sewituwgnoj. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-021-82928-4.. antigen (ABRA) or merozoite surface protein 9 (PfMSP9) forms a co-ligand complex with the 19?kDa fragment of merozoite surface protein 1 (MSP119) that binds to erythrocyte band 3 during the process of erythrocyte recognition/invasion2C4. PfMSP9 is usually peripherally associated with the merozoite surface and is found within the parasitophorous vacuole of MSP9 homologue has conferred protection against the parasite challenge11. Furthermore, antibodies to MSP9 (PvMSP9) were associated with protection against symptomatic infections among children in Papua New Guinea12, supporting the role of MSP9 as a promising vaccine candidate. Structural organization of PvMSP9 was similar to the orthologues in and other simian malaria species13, characterized by (1) the N-terminal signal peptide, (2) conserved cysteine residues at the N-terminal part and (3) repetitive amino acid sequences intervening conserved domains in which the locations of repeats seem to be variable across species14. It is noteworthy that this N-terminal conserved domain name I of PvMSP9 shared 82% and 77% amino acid sequence identity with its orthologs in (PcyMSP9) and (PkMSP9), respectively (Supplemental Fig. S1), rendering antigenic cross-reactivity among these malaria species9,13. Meanwhile, analysis of the complete PvMSP9 sequences from Thai field isolates TCF3 has revealed a relatively short repeat region in the middle portion of the protein whereas two other repeat domains occupied the majority of the C-terminal region. Extensive length and sequence diversity occurred mainly in the repeat domains of PvMSP9 among field isolates15 (Fig.?1). Open in a separate window Physique 1 (A) Structure of PvMSP9 depicting conserved domains (ICIII) and repeat domains (R1CR3). Approximate locations, amino acid substitutions and length of repeat domains in the recombinant proteins are shown correspondingly underneath. Four conserved cysteines at residues 49, 52, 64 and 71 Empagliflozin are marked with downward arrows and broken vertical lines in the upper diagram. Positions of the first Empagliflozin and the last Empagliflozin amino acid residues for Empagliflozin each recombinant protein are shown underneath at both ends of the schemes (B) Amino acid sequences of repeat domains R2 and R3 in recombinant proteins CT1750 and CT1756. The nomenclatures for the repeat units are listed above and below the sequences with corresponding colors. (C) Coomassie brilliant blue-stained SDS gel of purified PvMSP9 recombinant proteins. Lane M represents molecular weight marker. Lanes 1C4 are proteins TMS102, CT1186, CT1750 and CT1756, respectively, under reducing condition (with -mercaptoethanol). Lanes 5C8 are the corresponding proteins under non-reducing condition (without -mercaptoethanol). Like PfMSP9, both N- and C-terminal domains of PvMSP9 were Empagliflozin immunogenic upon natural infection16C18. To further address whether sequence variation in PvMSP9 could affect the profile of IgG antibody responses during natural contamination, we analyzed serum samples from malaria patients in Thailand against recombinant peptides derived from the N- and the C-terminal variants of this protein. Results revealed that most sera seem to recognize common or cross-reactive epitopes while some patients developed variant-specific antibodies. The majority of anti-PvMSP9 IgG antibodies were cytophilic isotypes while the levels and avidity of antibodies seems to be correlated with previous malaria episodes. Results Recombinant PvMSP9 proteins The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified recombinant proteins from the N-terminal a part of PvMSP-9 (TMS102 and CT1186) measured approximately 39?kDa, corresponding to the expected molecular mass. No shift in the migration pattern was observed for the N-terminal antigens under reducing and nonreducing SDS-PAGE, suggesting that this.