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Cloning, molecular characterization, and functional activity of Schistosoma japonicum glyceraldehyde-3-phosphate dehydrogenase, a putative vaccine candidate against schistosomiasis japonica.

ABSTRACT: We report the cloning, molecular characterization, and purification of functionally active recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the human bloodfluke Schistosoma japonicum. The GAPDH homolog from the related species Schistosoma mansoni has shown correlation of antibody titer to resistance to reinfection. A 1,164-bp cDNA (C1) was isolated from an S. japonicum lambda ZapII cDNA expression library immunoscreened with hyperimmune rabbit serum raised against soluble adult S. japonicum proteins. The open reading frame of C1 encodes a protein of 338 amino acids exhibiting 90% identity to the amino acid sequence of S. mansoni GAPDH. The inferred molecular mass of the protein is 36,589 daltons, and in vitro translation of the cDNA with [35S]methionine produced a radiolabelled band of the predicted size. Antibodies to C1 selected from hyperimmune rabbit serum by affinity purification recognized an S. japonicum protein doublet of 37 kDa but did not cross-react with a corresponding protein in S. mansoni extracts. The S. japonicum GADPH appears to be translated from a single mRNA encoded by a single-copy gene. After subcloning in the QIAexpress vector pQE-10 and subsequent expression, the recombinant protein was purified under nondenaturing conditions and shown to exhibit functional GAPDH enzymatic activity.

SUBMITTER: Waine GJ

PROVIDER: S-EPMC281226 | biostudies-other | 1993 Nov

REPOSITORIES: biostudies-other

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Cloning, molecular characterization, and functional activity of Schistosoma japonicum glyceraldehyde-3-phosphate dehydrogenase, a putative vaccine candidate against schistosomiasis japonica.

Waine G J GJ Becker M M Yang W W Kalinna B B McManus D P DP

Infection and immunity 19931101 11

We report the cloning, molecular characterization, and purification of functionally active recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the human bloodfluke Schistosoma japonicum. The GAPDH homolog from the related species Schistosoma mansoni has shown correlation of antibody titer to resistance to reinfection. A 1,164-bp cDNA (C1) was isolated from an S. japonicum lambda ZapII cDNA expression library immunoscreened with hyperimmune rabbit serum raised against soluble adult ...[more]

PMID: 8406870

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Project description:In recent years, the prevalence and infection intensity of Schistosoma japonicum in endemic areas of the Philippines have significantly decreased due to yearly population-based treatment strategies, yet transmission rates remain high and uninterrupted. An important indicator of active disease transmission is the presence of Schistosoma japonicum and its snail intermediate host Oncomelania hupensis quadrasi in freshwater habitats. In this study, we sought to apply a species-specific real-time PCR (qPCR) assay for the detection of S. japonicum and O. hupensis quadrasi in freshwater samples using environmental DNA approach that can complement the commonly utilized malacological survey in determining potential transmission foci in order to have a more effective snail surveillance strategy for schistosomiasis japonica in endemic areas. The newly developed assay was specific to S. japonicum and O. hupensis quadrasi with no amplification detected against non-target trematode Fasciola spp. and snails such as Lymnaea spp., Pomacea canaliculata, and Melanoides spp. that typically co-exist in the same environment. The assay effectiveness was determined using 19 environmental water samples collected from Northern Samar (N = 5 sites), Leyte (N = 11 sites) and Compostela Valley (N = 3 sites) and compared to malacological survey for determining O. hupensis quadrasi snail colonies and snail crushing to visualize S. japonicum cercariae. TaqMan qPCR targeting a short fragment of the cytochrome c oxidase subunit 1 (cox1) gene was positive for S. japonicum in 9 sites, for O. hupensis quadrasi in 9 sites, and for both S. japonicum and O. hupensis quadrasi in 5 sampling sites. Moreover, it was able to detect O. hupensis quadrasi in 3 out of 12 sites found negative and 6 out of 7 sites found positive through malacological survey, and in 4 of the 5 snail sites positive for snails with cercariae. Overall, this method can complement malacological surveys for monitoring of schistosomes in endemic areas of the Philippines, especially those with high risk of human infection.

Project description:BACKGROUND: Thyroid hormones (TH) modulate growth, development and differentiation and metabolic processes by interacting with thyroid hormone receptors (THRs). The purpose of this study was to identify a novel thyroid hormone receptor beta encoding gene of Schistosoma japonicum (SjTHR?) and to investigate its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice. METHODS: The full-length cDNA sequence of SjTHR?, its gene organization, and its transcript levels were characterized, and the phylogenetic relationship between THR, RAR and RXR from other organisms were analysis, the ability of this protein binding to a conserved DNA core motif, and its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice were evaluated. RESULTS: The SjTHR? cDNA was cloned, verified by 5' and 3' Rapid Amplification of cDNA Ends and shown to be polyadenylated at the 3'end, suggesting the transcript is full-length. SjTHR? is homologous to THRs from other species and has a predicted conservative DNA binding domain and ligand binding domain that normally characterizes these receptors. A comparative quantitative PCR analysis showed that SjTHR? was the highest expressed in 21d worms and the lowest in 7 d and 13 d schistosomula. The cDNA corresponding to DNA binding domain (SjTHR?-DBD) and ligand binding domain (SjTHR?-LBD) were cloned and subsequently expressed in E coli. The expressed proteins were used to immunize mice and generate specific serum against recombinant SjTHR? (rSjTHR?). Western blotting revealed that anti-rSjTHR?-LBD serum recognized two protein bands in extracts from 21 d worm with molecular sizes of approximately 95 kDa and 72 kDa. Electrophoretic mobility shift assay (EMSA) analysis showed that rSjTHR?-DBD could bind to a conserved DNA core motif. Immunization of BALB/c mice with rSjTHR?-LBD could induce partial protective efficacy(27.52% worm reduction and 29.50% liver eggs reduction)against schistosome infection. Enzyme-linked immunosorbent assay showed that mice vaccinated with recombinant SjTHR?-LBD (rSjTHR?-LBD) generated increased levels of specific IgG, IgG1 and IgG2a antibody. Bio-plex analysis demonstrated that rSjTHR?-LBD induced considerably higher levels of T helper 1 cytokines (IL-2, IL-12 and TNF-?) than T helper 2 cytokines (IL-10, IL-4), suggesting that rSjTHR?-LBD vaccination could stimulate mixed Th1/Th2 types with Th1 dominant immune responses. CONCLUSIONS: Our study presented here identified SjTHR? as a new schistosome THR that might play an important role in host-parasite interaction and be a vaccine candidate for schistosomiasis.

Dataset Information

Cloning, molecular characterization, and functional activity of Schistosoma japonicum glyceraldehyde-3-phosphate dehydrogenase, a putative vaccine candidate against schistosomiasis japonica.

Publications

Cloning, molecular characterization, and functional activity of Schistosoma japonicum glyceraldehyde-3-phosphate dehydrogenase, a putative vaccine candidate against schistosomiasis japonica.

Similar Datasets

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