Cloning, expression, and preliminary characterization of the dysferlin tegument protein in Schistosoma japonicum.

The schistosomal tegument is a dynamic host-interactive layer. Proteins exposed to the host on the tegumental surface are important for completion of the parasitic lifecycle. Dysferlin is a member of the ferlin family and is involved in plasma membrane repair. Based on the results of a proteomics study of tegument surface proteins of Schistosoma japonicum in our laboratory, dysferlin was identified as a tegumental protein of S. japonicum. The gene encoding S. japonicum dysferlin (SjDF), which codes for several Ca(2+) binding sites, was cloned, expressed in Escherichia coli, and characterized. Western blot analysis revealed that recombinant SjDF had good immunogenicity. Real-time RT-PCR analysis showed that SjDF was upregulated mainly in adult worms and the transcription level in 42-day-old female worms was significantly higher than that in males. Immunofluorescence analysis revealed that SjDF was mainly distributed in the tegument at various developmental stages. Experimental mice were treated with praziquantel and at 35days post-infection, we noted that damage to the tegument and subtegument worsened and did not recover at 36h post-treatment in the high-dose group and was accompanied by downregulation of SjDF mRNA, while the damage was less severe and recovered by this time in the low-dose group, and accompanied by upregulation of SjDF. Our results suggested that SjDF is a tegumental protein that may be important in schistosomal development and may participate in the repair process in muscle and tegument, and could present a viable vaccine candidate for schistosomiasis.

Characterization Analysis of Schistosoma japonicum Plasma Membrane Repair Relative Gene Myoferlin.

Myoferlin is a member of the ferlin family of proteins, which are involved in plasma membrane repair, and has been identified as one of the tegument proteins of Schistosoma japonicum. The tegument proteins are potential candidates for vaccines and new drug targets. In this study, myoferlin of S. japonicum (SjMF) was cloned, expressed and characterized, the potential of SjMF recombinant protein (rSjMF) as a vaccine candidate was evaluated, and the effect of praziquantel on SjMF was detected by Real-time PCR. Immunofluorescence showed that this protein was mainly distributed on the surface of worms at different stages. Sequence analysis revealed that the SjMF open reading frame was conserved at all stages of the S. japonicum life cycle. And SjMF transcription was upregulated in 42-day-old worms, and was significantly higher in female worms. Western blotting revealed that rSjMF showed strong immunogenicity. The cytokine profile and IgG isotype analysis demonstrated that rSjMF plus ISA206 immunization induced a mixed T helper (Th)1/Th2 response. Purified rSjMF emulsified with ISA206 adjuvant significantly reduced worm burden from 21.8% to 23.21% and liver egg number from42.58% to 28.35%. Besides, SjMF transcription was downregulated when worms were exposed to low-dose praziquantel (PZQ) and upregulated when PZQ was degraded, accompanied by recovery of damaged tegument. When worms were exposed to high-dose PZQ, SjMF transcription was downregulated all the time and the damaged tegument did not recover. These findings indicated that SjMF is a potential vaccine against S. japonicum and provides the basis for further investigations into the biological function of SjMF.

Gene gun bombardment with DNA-coated golden particles enhanced the protective effect of a DNA vaccine based on thioredoxin glutathione reductase of Schistosoma japonicum.

Schistosomiasis, caused by infection with Schistosoma species, remains an important parasitic zoonosis. Thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR) plays an important role in the development of the parasite and for its survival. Here we present a recombinant plasmid DNA vaccine, pVAX1/SjTGR, to estimate its protection against S. japonicum in BALB/c mice. The DNA vaccine administrated by particle bombardment induced higher protection than by intramuscular injection. All animals vaccinated with pVAX1/SjTGR developed significant specific anti-SjTGR antibodies than control groups. Moreover, animals immunized by gene gun exhibited a splenocyte proliferative response, with an increase in IFN- γ and IL-4. The recombinant plasmid administrated by gene gun achieved a medium protective efficacy of 27.83-38.83% (P < 0.01) of worm reduction and 40.38-44.51% (P < 0.01) of liver egg count reduction. It suggests that different modes of administering a DNA vaccine can influence the protective efficacy induced by the vaccine. Interestingly, from the enzymatic activity results, we found that worms obtained from pVAX1/SjTGR-vaccinated animals expressed lower enzymatic activity than the control group and the antibodies weakened the enzymatic activity of SjTGR in vitro, too. It implies that the high-level antibodies may contribute to the protective effects.

[Characterization and immunoprotective effect of SjIrV1, a 66 kDa calcium-binding protein from Schistosoma japonicum].

Calcium-binding protein is an indispensable protein which performs extensive and important functions in the growth of Schistosoma japonicum. Based on our primary study on tegument surface proteins of S. japonicun, a cDNA encoding a 66 kDa calcium-binding protein of S. japonicum (Chinese strain) was cloned, sequence analysis revealed that it was identical with that of SjIrV1 of Philippines strains S. japonicum. The expression of SjIrV1 were detected by Real-time PCR, using cDNA templates isolated from 7, 14, 21, 28, 35 and 42 days worms and the results revealed that the gene was expressed in all investigated stages, and the mRNA level of SjIrV1 is much higher in 42 d female worms than that in 42 d male worms. The cDNA containing the open reading frame of IrV1 was subcloned into a pET28a (+) vector and transformed into competent Escherichia coli BL21 for expression. The recombinant protein was purified using a Ni-NTA purification system, and confirmed by high performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS). Western blotting analysis showed that recombinant SjIrV1 (rSjIrV1) could be recognized by the S. japonicum infected mouse serum and the mouse serum specific to rSjIrV1, respectively. Immunofluorescence observation exhibited that SjIrV1 was mainly distributed on the tegument of the 35-day adult worms. ELISA test revealed that IgG, IgG1 and IgG2a antibodies are significantly increased in the serum of rSjIrV1 vaccinated mice. The study suggested that rSjIrV1 might play an important role in the development of S. japonicum.

Molecular characterization of thyroid hormone receptor beta from Schistosoma japonicum and assessment of its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice.

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 (SjTHRß) and to investigate its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice. METHODS: The full-length cDNA sequence of SjTHRß, 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 SjTHRß 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. SjTHRß 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 SjTHRß was the highest expressed in 21d worms and the lowest in 7 d and 13 d schistosomula. The cDNA corresponding to DNA binding domain (SjTHRß-DBD) and ligand binding domain (SjTHRß-LBD) were cloned and subsequently expressed in E coli. The expressed proteins were used to immunize mice and generate specific serum against recombinant SjTHRß (rSjTHRß). Western blotting revealed that anti-rSjTHRß-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 rSjTHRß-DBD could bind to a conserved DNA core motif. Immunization of BALB/c mice with rSjTHRß-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 SjTHRß-LBD (rSjTHRß-LBD) generated increased levels of specific IgG, IgG1 and IgG2a antibody. Bio-plex analysis demonstrated that rSjTHRß-LBD induced considerably higher levels of T helper 1 cytokines (IL-2, IL-12 and TNF-α) than T helper 2 cytokines (IL-10, IL-4), suggesting that rSjTHRß-LBD vaccination could stimulate mixed Th1/Th2 types with Th1 dominant immune responses. CONCLUSIONS: Our study presented here identified SjTHRß as a new schistosome THR that might play an important role in host-parasite interaction and be a vaccine candidate for schistosomiasis.

[Identification and preliminary analysis of a novel full-length cDNA encoding retinoid X receptor 2 from Schistosoma japonicum].

OBJECTIVE: To clone and preliminarily analyze the full-length cDNA encoding retinoid X receptor 2 (RXR2) from Schistosoma japonicum. METHODS: The rapid amplification cDNA ends (RACE)was applied to get a full-length cDNA encoding retinoid X receptor 2 from S. japonicum (SjRXR2). The transcription of SjRXR2 was detected by real-time PCR. By bioinformatical technology, the gene structure was analyzed and the antibody epitope was predicted. The polyclonal antibodies were raised in mice immunized with the synthesis peptide. Western blot was applied to detect its expression in the worm. RESULTS: The full-length cDNA of SjRXR2 was 5 960 bp and contained an open reading frame encoding a 1 435 amino acid which had a predicted molecular weight 159 kDa. Bioinformatical analysis indicated that SjRXR2 had a highly conserved DNA binding domain (DBD) and a moderate conserved ligand binding domain (LBD). The relative mRNA (s) of SjRXR2 with higher expressions at Day 21 and 42 were evaluated in five different S. japonicum developmental stages. The Western blot analysis showed that polyclonal antibodies were able to specifically recognize the protein with molecular around 150 kDa from the extract of S. japonicum. CONCLUSION: A full-length cDNA encoding retinoid X receptor 2 (RXR2) from S. japonicum is obtained which provides preliminary information for further investigation of SjRXR2 functions in S. japonicum.