Figure 1.
Protein sequence alignment of Schistosoma japonicum tetraspanin 2 (Sj-TSP-2) isolated from adult worms.
The variable region is boxed. The large extracellular loop region is indicated by a solid line. Sj-TSP-2a (M8-1 and M6-1, GenBank accession numbers JF264973 and JF264974), Sj-TSP-2c (M2-11, JF264975), Sj-TSP-2d (M9-1, JF264977), Sj-TSP-2e (F2-2, JF264978), Sj-TSP-2f (F12-1, JF264982; F11-4, JF264983), Sj-TSP-2h (F11-7, JF264979; F11-9, JF264980; F3-1, JF264981) and Sj-TSP-2i (M2-1, JF264976) were from individual male (M) and female (F) worms. For instance, M8-1 represents number 8 male worm and number 1 clone.
Table 1.
Subclass distribution of S. japonicum tetraspanin 2 (Sj-TSP-2) in male and female worms.
Figure 2.
Phylogenetic analysis of Sj-TSP-2 clusters with homologues.
Protein sequences corresponding to the LEL region of Sj-TSP-2 encoded by 9 clusters of Sj-TSP-2 cDNAs were aligned with additional homologues found by BLAST searches in GenBank and others reported by Cai et al [8]. Analysis was done as stated in the text. Clade credibility (posterior probability) values are shown at nodes. Protein sequence data reported in this paper are available in the GenBank, EMBL and DDBJ databases under the accession numbers ABR27733, AAX26611 and CAX70617.
Figure 3.
Expression levels of Sj-TSP-2e by real-time PCR.
cDNAs were amplified with mRNA isolated from different stages of S. japonicum using specific primers designed from the conserved regions of Sj-TSP-2e. Ce, cercariae; So, schistosomula; Pw, paired adult worms; Ma, males; Fe, females. The bars (and *, X axis) show the fold changes compared with the cercarial stage. We used NADH-ubiquinone reductase as a house-keeping gene to calculate the number of copies of the gene expressed in each of the stages, and then converted these to fold changes by comparison with the number of copies in cercariae.
Figure 4.
Purification and recognition of recombinant Sj-TSP-2e.
Left panel, SDS-PAGE of soluble and insoluble proteins extracted from adult S. japonicum and recombinant proteins. Right panel: Recognition of recombinant Sj-TSP-2e and native proteins by antibodies in hyper immune mouse serum raised against recombinant Sj-TSP-2e. Lane M, protein markers; Lanes 1 and 2, soluble and insoluble proteins of S. japonicum; lane 3, Thi; lane 4, Sj-TSP-2e-Thi; lane 5, Sj-TSP-2e expressed in Pichia yeast; lane 6, Sm-TSP-2e-Thi; lane 7, Sj-23-GST-His as a control protein.
Figure 5.
Recognition of recombinant Sj-TSP-2e by human sera.
I: ELISA IgG screen of 72 sera collected from positive schistosomiasis japonica patients and 24 normal human sera (negative controls) probed by protein preparation from S. japonicum adult worms (SWAP), recombinant tag protein thioredoxin (Thio) and Sj-TSP-2e. II. Panel a: SDS-PAGE of soluble and insoluble protein extracted from adult S. japonicum and recombinant Sj-TSP-2e. Panels b and c: Western blot analysis with pooled sera randomly selected from confirmed schistosomiasis japonica patients (n = 15) and pooled sera from negative control subjects (n = 15) from northern China. Lane M, protein markers; lanes 1 and lane 2, soluble and insoluble proteins from adult S. japonicum; lane 3, Sj-TSP-2e (arrowed); lane 4, thioredoxin tag (Thi) (arrowed); lane 5, Sj-TSP-2e expressed in Pichia yeast (arrowed).
Figure 6.
Immunolocalization of S. japonicum Sj-TSP-2e in adult female worms.
Parasite sections were reacted with specific antibodies produced in mice against recombinant Sj-TSP-2e. The antibodies that specifically bound to the sections were probed with an anti-mouse IgG labelled with Cy3 conjugate (a, c). Red fluorescence in panel c indicates Sj-TSP-2e is located in the parasite tegument (teg); anti-thioredoxin (fusion protein tagged with 6His) antibodies in panel a did not react. DAPI to label nuclei (nuc in blue) (b, d) was used as a quality control marker for the sections. Tegument, teg.
Figure 7.
Antibody isotype levels in mice challenged with 35 S. japonicum cercariae.
Mouse serum anti-Sj-TSP-2e (fused with thioredoxin, Thi) antibodies were determined by ELISA after primary vaccination with S. japonicum-TSP-2e (Sj-TSP-2e) or S. mansoni-TSP-2 (Sm-TSP-2). Thioredoxin was used as control protein in the vaccine trials. The thin arrows indicate vaccination time points and the bold arrows indicate the time point of cercarial challenge.
Table 2.
Vaccination results of mice vaccinated with SjTSP-2e against challenge infection with Schistosoma japonicum cercariae.