Figure 1.
Microscopical examination of C. sinensis metacercariae for cyst wall proteins.
(A) Intact metacercaria (×400); (B) Excysted juvenile, juvenile immediately moved out (×100); (C) Cyst wall (×400). Bar = 0.1 mm.
Table 1.
Proteins from cyst wall of C. sinensis metacercariae identified by HPLC-MS/MS.
Figure 2.
Multiple sequence alignment of deduced amino acid sequence of paramyosin among helminthes.
C. s-1 (JQ041818) represents the sequence from our C. sinensis metacercaria cDNA plasmid library. C. s-2 (ABN79674.1) represents the sequence submitted by the laboratory from Korea. Paragonimus westermani (P. w, AAY44740.1), Schistosoma haematobium (S. h, BAF62291.1), Schistosoma japonicum (S. j, AAA81003.1), Schistosoma mansoni (S. m, AAA29915.1), Taenia solium (T. s, AAK58494.1) and Echinococcus granulosus (E. g, CAA79849.1). Amino acids shared among helminthes were indicated in black, high conserved amino acids among helminthes were indicated in gray. B-cell and T-cell linear epitopes were indicated with full lines and dotted lines, respectively.
Figure 3.
Expression and purification of recombinant pET-26b-CsPmy identified by 8% SDS-PAGE.
(A) Expression of pET-26b-CsPmy. Protein molecular weight marker (M), lysate of E. coli with pET-26b(+) before induction with IPTG (lane 1) and after induction (lane 2), lysate of E. coli with pET-26b-CsPmy before induction with IPTG (lane 3) and after induction (lane 4), supernatant of induced E. coli with pET-26b-CsPmy (lane 5) and sediment (lane 6). (B) Denaturation of inclusion bodies containing pET-26b-CsPmy. Supernatant collected from inclusion bodies dissolved in 2 M urea (lane 1) and 6 M urea (lane 2). (C) Purification of pET-26b-CsPmy. Protein eluted with 40 mM imidazole (lane 1–3), 80 mM imidazole (lane 4–8), 100 mM imidazole (lane 9–12), 200 mM imidazole (lane 13–14). Proteins were visualized by Coomassie Blue staining, the protein bands were around 100 kDa.
Figure 4.
Identification of CsPmy by SDS-PAGE and Western blot analysis.
Protein molecular weight marker (M), purified pET-26b-CsPmy protein (lane 1), TWE of adult worm (lane 2), TWE of metacercaria (lane 3), TWE of cercaria (lane 4), TWE of egg (lane 5), cyst wall proteins of metacercaria (lane 6), and soluble tegumental components of adult worm (lane 7). (A) 8% SDS-PAGE. (B) Western blot analysis. Corresponding proteins were subjected to 8% SDS-PAGE and immobilized onto the membrane, then the membrane was incubated with anti-pET-26b-CsPmy rat serum (1∶2000 dilutions) at room temperature for 2 h. Subsequently, the membrane was followed by incubation with rabbit anti-rat IgG HRP-conjugated secondary antibody (1∶2000 dilutions) at room temperature for 1 h. 2 µg of purified pET-26b-CsPmy protein and 10 µg of TWE were loaded per lane. SDS-PAGE was visualized by Coomassie Blues staining and the protein bands that might be native paramyosin in different life stages were indicated with arrows. Western blot was visualized by ECL method, the detected protein bands were around 100 kDa.
Figure 5.
Transcriptional level of CsPmy at different developmental stages of C. sinensis by qRT-PCR experiments.
Total RNA from four stages (adult worm, metacercaria, cercaria and egg) were extracted by TRIzol methods and spectrophotometrically quantitated. Reverse transcription reactions were carried out to get the first-strand cDNA with the same quantity of total RNA as the template (1 µg). β-actin of C. sinensis (accession number: EU109284) was used as the transcription control. The real-time PCR amplification was performed using the LightCycler480 instrument (Roche, Switzerland) using the SYBR Premix ExTaq Kit. The LightCycler480 software (version 1.5) was used to analyze the data according to the 2−ΔΔCt method [27]. The amplification of egg was employed as the calibrator to evaluate relative expression levels of CsPmy.
Figure 6.
Immunohistochemical localization of CsPmy at adult worm and metacercaria.
Adult worms and metacercariae of C. sinensis were fixed with 4% paraformaldehyde, embedded with paraffin and sliced into 3–5 µm in thick. The sections were blocked with normal goat serum overnight at 4°C, and then incubated with primary antibody (1∶200 dilutions) at room temperature for 2 h. After washing procedures, the sections were incubated with goat anti-rat IgG Alexa Fluor 594 (1∶400 dilutions) at room temperature for 1 h in dark. The images were captured under fluorescence microscope (ZEISS, Goettingen, Germany). Panel A–H, adult worm of C. sinensis. Pane I–L, metacercariae of C. sinensis. Pane A, B, E, F, I and J were sections treated with anti-pET26b-CsPmy serum. C, D, G, H, K and L were sections treated with naïve serum and imaged under the same conditions. Specific immunofluorescence was indicated in red (pane A, E and I), while no immunofluorescence was detected in pane C, G and K. Corresponding white light of parasite was panel B, D, F, H, J and L. T, tegument. OS, oral sucker. V, vitellarium. CW, cyst wall. EB, excretory bladder. Magnification for adult worm and metacercaria were ×100 and ×400, respectively.
Figure 7.
Antibody titers of IgG induced by CsPmy measured by ELISA.
Briefly, 1 µg/well recombinant pET-26b-CsPmy protein was coated on the plates and blocked with 5% skimmed milk. The plate was incubated with different dilutions of the immune sera (week 6) raised by pET-26b-CsPmy and pcDNA-CsPmy. Rat sera immunized with PBS and pcDNA were measured under the same conditions as negative controls. HRP-conjugated IgG (1∶20000 dilutions) was used as the secondary antibodies. The reactions were developed with substrate solution TMB, stopped by 2 M H2SO4 and measured at measured at 450 nm. (A) Antibody titers of IgG induced by pET-26b-CsPmy. (B) Antibody titers of IgG induced by pcDNA-CsPmy.
Figure 8.
IgG isotype induced by CsPmy measured by ELISA.
1 µg/well recombinant pET-26b-CsPmy protein was coated on the plates and blocked with 5% skimmed milk. Immune sera from week 2 to week 6 were diluted at 1∶400. Rat sera immunized with PBS and pcDNA were measured under the same conditions as negative controls. IgG (1∶20000 dilutions), IgG1 and IgG2a (1∶1000 dilutions) were used as secondary antibodies. (A) Immune responses induced by pET-26b-CsPmy. (B) Immune responses induced by pcDNA-CsPmy.
Table 2.
Protective effect of in vaccination trialsa.