Sequence conservation of P. vivax glutamate dehydrogenase among Korean isolates and its usefulness in seroepidemiology

초록

Background: The glutamate dehydrogenase of malaria parasites (pGLDH) is widely used in a rapid diagnostic test. Variation in the pGLDH gene among Korean isolates was analyzed, and a recombinant pGLDH protein was evaluated as a serological antigen of vivax malaria. Methods: Genomic DNA was purified and the pGLDH gene of Plasmodium vivax was sequenced from the blood samples of 20 patients. Recombinant protein was prepared to determine the antigenicity of pGLDH as tested by enzyme-linked immunosorbent assay. Results: Partial sequence analysis of the P. vivax pGLDH gene from the 20 Korean isolates showed that the open reading frame of 1,410 nucleotides encoded a deduced protein of 470 amino acids. The amino acid and nucleotide sequences were conserved among all the Korean isolates. This ORF showed 99.6% homology with P. vivax strain Sal-I (XP_001616667). The full ORF (amino acids 39?503), excluding the region before the intron, was cloned from the sequence of isolate P. vivax Bucheon 3 (KJ726751) and subcloned into the expression vector pET28b for transformation into Escherichia coli BL21(DE3)-pLysS. The expressed recombinant protein had a molecular mass of approximately 55 kDa, and the recombinant pGLDH showed 84.8% sensitivity (39/46 cases) and 97.2% specificity (35/36 cases) in an ELISA. To evaluate efficacy of pGLDH recombinant protein in sero-epidemiological study, it was adopted in ELISA. Blood samples collecting from 876 inhabitants who lived in Gyodong-myeon, Ganghwa-county, Incheon Metropolitan city, were tested, among them, 91 persons (10.39%) showed in positive reaction with pGLDH recombinant protein. Among antibody positive cases, 13 cases (14.29%) have an experience of malaria infection during last ten years. Conclusion: The pGLDH genes of P. vivax isolates from representative epidemic areas of South Korea were highly conserved, without change of nucleotides.