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Speciation plays a central role in evolutionary studies, and particularly how reproductive isolation (RI) evolves. The origins and persistence of RI are distinct processes that require separate evaluations. Treating them separately clarifies the drivers of speciation and then it is possible to link the processes to understand large-scale patterns of diversity. Recent genomic studies have focused predominantly on how species or RI originate. However, we know little about how species persist in face of gene flow. Here, we evaluate a contact zone of two closely related toad-headed lizards (Phrynocephalus) using a chromosome-level genome assembly and population genomics. To some extent, recent asymmetric introgression from Phrynocephalus putjatai to P. vlangalii reduces their genomic differences. However, their highly divergent regions (HDRs) have heterogeneous distributions across the genomes. Functional gene annotation indicates that many genes within HDRs are involved in reproduction and RI. Compared with allopatric populations, contact areas exhibit recent divergent selection on the HDRs and a lower population recombination rate. Taken together, this implies that divergent selection and low genetic recombination help maintain RI. This study provides insights into the genomic mechanisms that drive RI and two species persistence in the face of gene flow during the late stage of speciation.

Background This study investigated the effect of flight transport stress on beagles' routine blood indexes and biochemical parameters and evaluated the anti‐stress effect of dangshen (Codonopsis pilosula). Methods We selected 12 beagles and divided them into two groups. One group was treated with dangshen decoction two hours before the flight, and the other group was untreated. Their routine blood indexes and clinical biochemical parameters were tested and analyzed before transport, after unloading and after adaptation for 1, 2, 3, 4, 5, and 6 days after administering dangshen. Results We found that flight transportation stress adversely influenced many of the beagles' routine blood indexes. These recovered during adaptation, with dangshen administration assisting recovery of most indexes. Flight transport stress also adversely influenced biochemical indexes in the beagles. Again these recovered during adaptation, and dangshen aided in the recovery. Conclusion Thus, we found that flight transport adversely affected the beagles' blood indexes, and dangshen reversed the damage from transport stress.

To study the immunogenicity and the immuno-protection of thioredoxin glutathione reductase from Schistosomajaponicum (SjTGR) against schistosome infection in mice.Seventy-five mice were randomly divided into 5 groups, namely, blank group, PBS group, CpG2 immunized group, TGR immunized group and TGR + CpG2 co-immunized group. Each mouse was immunized for 3 times. The mice were tail bled before the first immunization and 2 weeks after the third immunization. The serum antibody levels of total IgG, IgG1 and IgG2a against SjTGR were assayed by ELISA. Two weeks after the third immunization, each mouse was infected with 40 ± 2 S. japonicum cercariae by abdominal skin penetration. Forty-two days later, all the mice were sacrificed to collect schistosome adult worms and liver eggs. The worm and egg reduction rates were calculated respectively. The single splenocyte of mouse was collected 2 weeks after the third immunization, and the expressions of CD44high, CD4+CD44high or CD8+CD44high on splenocytes of mice were examined by flow cytometry. After 72 h incubation with recombinant SjTGR, the levels of IL-2, IL-4, IL-10, and IFN-γ in the single-cell supernatant were determined by using ELISA kit.Two weeks after the third immunization, the titers of serum IgG against SjTGR in mice immunized with SjTGR and co-immunized with SjTGR and CpG2 were higher than 1:200 000. The IgG2a: IgG1 ratio (IgG2a/IgG1) increased slowly with time in both TGR immunized group and TGR + CpG2 co-immunized group. There were obviously higher levels of IFN-γ and IL-2 in the cell supernatant in the TGR immunized group and TGR + CpG2 co-immunized group compared to the blank, PBS and CpG2 groups (P0.05). The increased subpopulations of CD44high, CD8+CD44high and CD4+ CD44high cells in the splenocytes from mice immunized by SjTGR and co-immunized by SjTGR and CpG2 were found comparing to the blank, PBS and CpG2 groups (P0.05). The TGR immunization and TGR + CpG2 co- immunization caused 9.4% and 10.5% reductions in the number of adult worms and 9.2% and 32.8% reductions in the number of eggs, respectively.SjTGR displays strong immunogenicity inducing Th1 type immune response in mice. However, it could not produce protective efficacy against S. japonicum infection. CpG2 ODN may be a broadly effective Th1 adjuvant.

Procedures for the preparation of compounds. (DOCX 36 kb)

Killing activity on juvenile S. japonicum in vitro by oxadiazole-2-oxides. (DOCX 27 kb)

Killing activity on adult S. japonicum in vitro by oxadiazole-2-oxides. (DOCX 23 kb)

Nitric Oxide (NO) production of oxadiazole-2-oxides with SjTGR+NADPH or with SjTGR alone, or with worm homogenate supernatant. (DOCX 17 kb)

To explore the performance of the biotin-avidin complex enzyme linked immunosorbent assay of detecting specific IgG4 for the diagnosis of clonorchiasis.The avidin-biotin complex enzyme linked immunosorbent assay of detecting specific IgG4 (IgG4-ABC-ELISA)against Clonorchis sinensis was established, and used to detect the serum samples of patients with clonorchiosis sinensis, schistosomiasis japonica, paragonimiasis, toxoplasmosis, echinococcosis, cysticercosis and sparganosis mansoni. At the same time, these sera were analyzed by the ELISA of detecting IgG4 (IgG4-ELISA) and ELISA of detecting the total IgG (IgG-ELISA) as controls. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the respective diagnostic performance of the three methods were compared.The IgG4-ABC-ELISA for diagnosis of clonorchiasis was established successfully. The sensitivity and specificity of the IgG4-ABC-ELISA for detecting clonorchiasis were 90.0% and 98.2% respectively, and PPV and NPV were 93.8% and 97.0% respectively. Its diagnostic performance was 96.3%. The sensitivity and specificity of the IgG4-ELISA for detecting clonorchiasis were 86.0% and 98.2% respectively, and PPV and NPV were 93.5% and 95.9% respectively. Its diagnostic performance was 95.4%. The sensitivity and specificity of the IgG-ELISA for detecting clonorchiasis were 94.0% and 88.1% respectively, and PPV and NPV were 70.1% and 98.0% respectively. Its diagnostic performance was 89.4%. The sensitivity of IgG4-ABC-ELISA was higher than that of IgG4-ELISA (P0.05), and the specificity of IgG4-ABC-ELISA was higher than that of IgG-ELISA (P0.05).IgG4-ABC-ELISA of detecting specific antibody IgG4 against Clonorchis sinensis has high sensitivity and specificity. Therefore, it has a good application value in the diagnosis of clonorchiasis.

Enolase is one kind of important glycolytic enzymes which widely exists in most organisms. A number of recent studies confirm that this enzyme has the functions of activating the plasminogen, involving in the processes of infection and migration of parasites, reducing the immune function of the host as well as preventing parasites from the immune attack of the host. This paper reviews the current research advances in the parasite enolase, and explores its potential for diagnosis, drug development and vaccine target of parasitic diseases.

To clone and express a high mobility group box 1(HMGB1) protein of Schistosomajaponicum (Mainland strain) and analyze its function.The DNA fragment of open reading frame encoding Sj HMGB 1 protein was amplified by RT-PCR from the mRNA of S. japonicum worms, then it was subcloned into the expression vector pET28a(+) to form the recombinant expression plasmid SjHMGB1-pET28a. The recombinant expression plasmid was transformed into the component E. coli BL21(DE3), and the tranformant containing recombinant expression plasmid was induced with IPTG to express the recombinant protein SjHMGB1. The recombinant SjHMGB1 protein was purified by affinity chromatography with nickel chelating affinity chromatography agarose gel. The Gel retard experiment and animal immunization were performed to analyze the DNA binding capacity and the immunologic property of recombinant SjHMGB1. The expression levels of HMGB1 in different life cycle stages of S. japonicum were analyzed by Western bloting and RT-PCR. Female ICR mice were immunized with the recombinant SjHMGB1 protein and infected with 45 +/- 2 cercariae of S. japonicum after three immunizations. Forty-two days post-infection, the worms and eggs of S. japonicum were recovered from the portal vein and liver tissue, respectively. The worm and egg reduction rates were calculated respectively.A 530 bp of specific DNA fragment was amplified from mRNA of S. japonicum by RT-PCR, which was the open reading frame (ORF) encoding SjHMGBlprotein confirmed by DNA sequencing analysis. The recombinant expression plasmid SjHMGB1-pET28a was constructed by cloning the ORF of SjHMGB1 into a expression vector pET28a(+). The bacterium transformants containing the recombinant plasmid expressed a soluble recombinant protein about 28 kDa after induced by IPTG, and the recombinant SjHMGB1 protein was purified by nickel chelating affinity chromatography. The gel retard experiment showed that the recombinant SjHMGB1 protein could bind to both supercoiled DNA and linear DNA, and the recombinant protein immunized mice produced high titers of antiserum IgG. Western bloting indicated that the recombinant SjHMGB1 protein was recognized specifically by the S. japonicum-infected mice serum. Above results showed that the recombinant SjHMGB1 protein possessed both functional activity and immunogenicity as the natural protein. RT-PCR and Western blot results showed that SjHMGB1 was abundantly expressed in the adult and egg stages whereas barely detectable in the cercaria stage. The immune protection experiment showed that the recombinant SjHMGB1 induced mice to produce high titers of specific antibody IgG but failed to conduct an effective immune protection against S. japonicum.The gene encoding HMGB1 from S. japonicum and the soluble recombinant SjHMGB1 protein with natural functional activity are obtained, and the recombinant SjHMGB1 has a high immunogenicity but is not able to induce an effective immune protection against S. japonicum.

CLAWN miniature pig has been shown to serve as a suitable host for the experimental infection of Schistosoma japonicum. In this study, we found that radiation-attenuated cercaria (RAC) vaccine gave CLAWN miniature pigs protective immunity against subsequent challenge infection with S. japonicum cercaria. To characterize the protective immune response of the pig model vaccinated by attenuated cercaria, flow cytometric analysis of the reactive T cell subsets was performed. The intracellular interferon (IFN)-γ and the cell surface markers revealed the peripheral blood CD3+ T-lymphocytes produced significant amounts of IFN-γ during the immunization period and after the challenge infection. CD4+ αβ-T cells as well as CD4+/CD8αmid double positive and/or CD8αhigh αβ-T cells were the major IFN-γ-producing CD3+ T cells. On the contrary, γδ T cells did not produce intracellular IFN-γ. Our results suggested that RAC-vaccinated miniature pigs showed effective protective immunity through the activation of αβ T cells bearing antigen specific T-cell receptors but not through the activation of γδ T cells., Tropical Medicine and Health, 42(4), pp.155-162; 2014

Antibody has extensive application prospects in the biomedical field. The inherent disadvantages of traditional polyclonal antibody and monoclonal antibody limit their application values. The humanized and fragmented antibody remodeling has given a rise to a series of genetic engineered antibody variant. This paper reviews the progress of research on genetic engineering antibody and its application in prevention and control of parasitic diseases.

Following the advancement in molecular biology and recombinant technology, the protein and polypeptide therapeutics have become an important class of clinical drugs due to their unique superiority. However, the problems of stability and short half-life of proteins and peptides also vastly limit their further use in clinic. The focal points of this paper are to introduce the specific ways and the matters needing attention to increase the biological half-life of the therapeutic protein and polypeptide drugs.

According to the requirements of the Mid- and Long-term Plan for Prevention and Control of Schistosomiasis in China and based on the actual situation of schistosomiasis control in Jiangsu Province, this paper demonstrates the new technologies achieved by the scientific innovation and the novel control strategies including integrated control in key regions and control of key populations and surveillance and forecast of key water regions since 2004, with the emphasis on the control and elimination of infected Oncomelania snails. Such strategies and technologies implemented the result in continuous decrease in the endemic situation of schistosomiasis in the province, and the whole province achieved the goal of schistosomiasis transmission control in late 2010.

Epitope is the basic functional motif of antigen inducing immune response. In immunological research and medical practice, the identification and selection of epitopes are the key points. This paper reviews the current progress of the epitope research and its application in disease diagnosis, vaccine research, disease treatment and other fields.

To identify the epitope of monoclonal antibody (McAb) 5C6 against 14-3-3 protein of Schistosoma japonicum by phage display peptide library.The phage display 12-peptide library was screened with purified McAb 5C6 against 14-3-3 protein of S. japonicum three rounds of bio-panning "adsorption-elution-amplification" to enrich the specific binding phages. The single phage clones selected randomly were amplified, their genomic DNA were extracted and sequenced. The immune response characterization of phages with the same or high homologous foreign inserted DNA sequence was identified by ELISA and Western blotting for further defining the epitope recognized by McAb 5C6.A total of 33 single phage clones were selected and sequenced. Among them, 25 shared the same foreign inserted DNA sequence of 5'-CCACCTAGTAGCAGACCGATTCTCAGTCGAAGGAAA-3', encoding a deduced peptide PPSSRPILSRRK. This peptide was not homologue to Sj14-3-3 protein or any other known native protein in the world. The results of Western blotting showed that this peptide could be recognized by the sera of patients with schistosomiasis, but not by those of healthy persons.The mimic antigen epitope of McAb 5C6 against 14-3-3 protein of S. japonicum, which is a conformational epitope, has been defined successfully in this study.

To prepare the fusion protein of large hydrophilic domain of 23 kDa membrane protein of Schistosoma japonicum and the mature peptide of human serum albumin (Sj23HD-HSA) and investigate its immunoreactivity.A fusion protein gene encoding Sj23HD-HSA fusion protein was prepared by overlapping PCR, which was confirmed by TA cloning and DNA sequencing. The fusion gene of Sj23HD-HSA was directionally subcloned into yeast expression plasmid pWX530 to construct a recombinant plasmid Sj23HD-HSA/pWX530. The transformants of Saccharomyces cerevisiae containing the recombinant plasmid Sj23HD-HSA/pWX530 were screened on leu deficient SD medium after yeast competent cells were transformed with recombinant plasmid. The excretive Sj23HD-HSA protein was expressed by culturing the yeast transformants at 30 degrees C for 1 week, and the protein component of culture supernatant was analyzed by SDS-PAGE. Sj23HD-HSA fusion protein was purified through Ion Exchange Chromatography. The immunoreactivity of recombinant Sj23HD-HSA fusion protein was determined by Western blotting with sera of schistosomiasis, clonorchiasis and healthy.The gene encoding the Sj23HD-HSA fusion protein was constructed successfully, which was confirmed by DNA sequencing. The yeast transformants containing plasmid Sj23HD-HSA/pWX530 could express the excretive Sj23HD-HSA fusion protein without inducing. The results of Western blotting indicated Sj23HD-HSA could be recognized by the sera of schistosomiasis, but could not be recognized by the sera of clonorchiasis and healthy respectively.Sj23HD-HSA fusion protein with good immune reactivity is prepared successfully, which will be a potential antigen for schistosomiasis immunodiagnosis.

To find out the candidate antigen for immunoreagent, which could be used to diagnose Schistosoma japonicum infection early in mice.The mice were infected with cereariae of S. japonicum Chinese mainland strain. The sera of mice before and after infection at different time were collected. The recombinant fusion protein (GST-HD) of the large hydrophilic domain (HD) of 23 kDa membrane protein of S. japonicum with the Glutathione-S-transferase (GST) of S. japonicum, soluble eggs antigen (SEA), TSP2 hydrophilic domain of S. japonicum (TSP2HD), IL4-inducing principle of S. mansoni eggs (IPSE), fusion protein GST-SjMP10 (SjMP-10), and recombinant S. japonicum (Chinese strain) signaling protein 14-3-3 (Sj14-3-3) were used as diagnostic antigens, the specific IgG and IgM antibodies were measured respectively by enzyme linked immunosorbent assay (ELISA). The antigens with the value of diagnosing schistosomiasis early were screened by analyzing the changes of the levels of specific IgG (or IgM) antibodies and the positive rates of specific antibodies in the sera of mice before and post infection at different time. Moreover, the antigen's value of early diagnosis was further validated by Immunoblot.On the 18th, 21st and 28th day post infection, the positive rates of specific antibody IgM against GST-HD were 60%, 70% and 100%, respectively; the positive rates of specific antibody IgG against GST-HD were 40%, 60% and 90%, respectively. The positive rates of antibody IgM against SEA were 50%, 60% and 90%, respectively; the positive rates of antibody IgG against SEA were 20%, 50% and 70%, respectively. The positive rates of IgM against TSP2HD were 30%, 40% and 50%, respectively; the rates of IgG against TSP2HD were 20%, 30% and 70%, respectively. The positive rates of IgM against IPSE were 20%, 30% and 50%, respectively; the positive rates of IgG against IPSE were 20%, 30% and 60%, respectively. The positive rates of IgM against SjMP-10 were 10%, 20% and 20%, respectively; the rates of IgG against SjMP-10 were 10%, 20% and 30%, respectively. The positive rates of IgM against Sj14-3-3 were 0, 10% and 20%, respectively; the rates of IgG against Sj14-3-3 were 0, 10% and 30%, respectively. The sensitivities of GST-HD and SEA for diagnosing schistosome infection early in mice were significantly higher than those of Sj14-3-3, IPSE, TSP, and MP-10. The sensitivity of IgM was higher than that of IgG. In Western blotting, the about 73 kDa protein band of SEA was recognized by sera of mice one week post infection and showed stronger reaction as the infected time went on. Moreover, the bands (33 kDa) of GST-HD were earliest recognized by the mouse sera on the 10th day post-infection, the bands showed strong reaction with the mouse sera of 5-week post-infection.The GST-HD fusion protein and the protein of which molecular weight is about 73 kDa of SEA have the early diagnostic value for schistosomiasis, and the sensitivity of Immunoblot is higher than that of ELISA.

To prepare the recombinant thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR) with biological activity.The open reading frame DNA sequence of SjTGR was fused with a bacterial-type selenosysteine insertion sequence (SECIS) element by PCR to form a chimeric gene. The chimeric gene was subcloned into expression plasmid pET-41a to construct a recombinant plasmid SjTGR-pET-41a. Then the recombinant plasmid SjTGR-pET-41a was co-transformed into E. coli BL21 with plasmid pSU ABC. The SjTGR protein was expressed by inducing with IPTG. The recombinant SjTGR was purified from expression products by affinity chromatography with an adenosine 2', 5'- diphosphate agarose column. The polyclonal anti-serum against recombinant SjTGR was obtained by immunizing mice with purified SjTGR. The native TGR in S. japonicum was evidenced by using Western blotting. Thiorendoxin reductase (TrxR) activity, glutathione reductase (GR) activity and gluaredoxin (Grx) activity of recombinant TGR were analyzed according to the biochemical method.The chimeric gene of SjTGR with a bacterial-type selenosysteine insertion sequence (SECIS) element was constructed successfully. The bacteria containing the recombinant plasmid SjTGR-pET-41a could express the soluble SjTGR by inducing with IPTG at static growth stage for 24 h at 24 degrees C. The expressed products of plasmid pSU ABC could promote the integration of selenocysteine and increase the yield of selenoprotein. The result of Western blotting showed that the polyclonal antiserum against recombinant SjTGR could recognize the native TGR in S. japonicum adult worms. The enzymatic assay indicated that SjTGR was a multifunctional enzyme with activities of TrxR, GR and Grx.The recombinant SjTGR with biological activity is expressed successfully, which lays the foundation for further study on the function and applied values of SjTGR.

To observe the toxicity of auranofin, cisplatin, adriamycin, compounds 4N, H, B, O against Schistosoma japonicum adult worms in vitro and their inhibition on thioredoxin glutathione reductase (TGR).The drugs mentioned above with different concentrations were added into RPMI 1640 medium with Schistosoma japonicum adult worms, which had been cultured for 30 - 60 min. The activity, morphological changes and death situation of the worms were observed after 1, 6, 24, 48 h and 72 h, respectively, then the worms were transferred to fresh medium without drugs to observe whether their activity would be recovered, and 50% lethal dose (LD50) of the drugs against adult worms was determined. The TrxR and GR activities of thioredoxin glutathione reductase of Schistosoma japonicum in homogenized supernatant of adult worms processed by drugs were tested following the DTNB reduction and NADPH oxidation methods.The mortality rates of 5 microg/ml of auranofin treating for 24 h, 20 microg/ml of 4N treating for 72 h, 60 microg/ml of H treating for 72 h, and 80 microg/ml of cisplatin treating for 72 h on adult worms were 100%, 60%, 66.7% and 100%, respectively, and there were statistically significant differences compared with the negative control group. LD50(s) of auranofin, 4N, H and cisplatin were 2.56, 17.59, 54.14 microg/ml and 52.87 microg/ml, respectively, but no toxic effects of other drugs on schistosome worms were found. The toxic effects of auranofin, 4N, cisplatin and H on adult worms were irreversible. Auranofin and cisplatin inhibited TGR activity of Schistosoma japonicum, but other drugs had no similar effect. 5 - 30 microg/ml of auranofin, 20 - 30 microg/ml of 4N, 70 - 150 g/ml of cisplatin, and 60 - 220 microg/ml of H caused the morphological changes of the worms after treating for 24 h.Auranofin, cisplatin and compounds 4N and H have toxicity on Schistosoma japonicum adult worms in vitro, and the schistosomicidal effect of auranofin and cisplatin may be related to the inhibition of TGR activity.

To optimize the experimental conditions of fast enzyme-linked immunosorbent assay (F-ELISA) and evaluate its performance for immunodiagnosis of schistosomiasis japonica.HRP-SPA was used at different concentrations in the assay to determine the best HRP-SPA concentration by comparing the detection accuracy. The working conditions of F-ELISA were optimized by examining the ratio of absorbance values of positive and negative controls with different incubation time of serum samples and HRP-SPA. To evaluate the performance of F-ELISA in diagnosis of schistosomiasis, the detection accuracy of F-ELISA was compared with routine ELISA and dipstick dye immunoassay (DDIA) by testing serum samples from both healthy subjects and those clinically diagnosed with schistosomiasis and clonorchiasis in parallel.In F-ELISA, the best working concentration of HRP-SPA was 1:2 000 while the optimized incubation condition for serum samples and HRP-SPA was 60 min at 37 degrees C. Using F-ELISA, we identified 93.8% to be schistosomiasis positive among chronic schistosomiasis subjects, 4% within the clonorchiasis group, and a false positive rate of 1.5% in healthy individuals. This diagnostic accuracy for F-ELISA was similar to routine ELISA and DDIA.F-ELISA combines the 2-steps of routine ELISA into a single step making the assay process faster and simpler without sacrificing the diagnostic accuracy. With further investigations, F-ELISA as an easy and practical method is promising to be applied for schistosomiasis diagnosis in the field.

To prepare soluble recombinant signal transduction protein 14-3-3 of Schistosoma japonicum (rSj14-3-3) and investigate its immunologic features.The cDNA fragment of signal transduction protein 14-3-3 gene was prepared from Schistosoma japonicum adult worm mRNA, and was subcloned to the downstream of glutathione S transferase gene of expression vector pGEX-4T-3 to construct a recombinant expression plasmid 14-3-3/pGEX-4T-3. The recombinant plasmids were transferred into E. coli BL21, the transforments containing recombinant plasmid were induced by IPTG, and the expression situation of fusion protein GST-14-3-3 was observed by SDS-PAGE. The pure recombinant 14-3-3 protein was prepared by digesting the fusion protein GST-rSj14-3-3 with thrombin and affinity chromatography. The specific antibody against rSj14-3-3 protein was prepared by an immunized rabbit with rSj14-3-3 protein. The immunogenicity and immune reactivity of rSj14-3-3 protein were analyzed by Western blotting.The gene encoding signal transduction protein 14-3-3 of Schistosoma japonicum Jiangsu strain was cloned successfully, the homology of open read frame sequence to the registrated sequence of Sj14-3-3 protein in genbank was 99.08%. A 55 kilo Dalton fusion protein GST-rSj14-3-3 was expressed by transferring the recombinant plasmid 14-3-3/pGEX-4T-3 into E. coli BL21 and induced with IPTG. The high titer antibody against rSj14-3-3 was produced by the immunized rabbit with rSj14-3-3 ,and could recognize the nature and recombinant rSj14-3-3 proteins.The soluble rSj14-3-3 protein has been prepared successfully in this study, and it has good immunogenicity and reactivity.

To identify the properties of six monoclonal antibodies (McAbs) against recombinant signal protein 14-3-3 of Schistosoma japonicum, and investigate their value in the diagnosis of schistosomiasis.The subclasses, titers, affinity-constants, detection limits and specificities of six McAbs were identified by ELISA and Western blotting. Dot Enzyme-linked Immunosorbent Assay (Dot-ELISA) for detecting the 14-3-3 protein in the sera of rabbits infected with Schistosoma japonicum was established, then it was used to observe the dynamics of 14-3-3 protein in sera of rabbits infected with Schistosoma japonicum before and after treatment. The diagnostic value of Dot-ELISA was investigated through detecting a group of sera samples of rabbits infected with Schistosoma japonicum.The types of six McAbs against recombinant signal protein 14-3-3 of Schistosoma japonicum of 3F1, 3F7, 5C6, 5D1, 5G9 and 1G6 were all IgG, their subclasses were IgG1, IgG2a, IgG2b, IgG1, IgG1 and IgG1, their antibody-titers in ascites were 1:6.4 x 10(5), 1:8.0 x 10(5) , 1:6.4 x 10(5), 1:3.2 x 10(5), 1:4.8 x 10(5) and 1:2.0 x 10(4), their affinity-constants were 8.82 x 10(8), 4.93 x 10(8), 1.56 x 10(8), 5.12 x 10(8), 1.41 x 10(8) moL/L and 2.30 x 10(7) mol/L, their detection limits in dot-ELISA were 1, 10, 100, 10, 10, 100 ng, respectively. All the six McAbs recognized the recombinant signal protein 14-3-3 of Schistosoma japonicum and the natural signal protein 14-3-3 in SEA, ESA and AWA, and did not react with the protein of E. coli and Clonorchis sinensis in Western blotting. 3F1 and 5D1 were selected to establish the Dot- ELISA for detecting the sera of rabbits infected with Schistosoma japonicum before and after treatment at different time points, it was found that the concentration of 14-3-3 protein in sera of rabbits was increased gradually with time and decreased gradually after the infected rabbits treated with praziquantel. Forty-two sera samples of rabbits infected with Schistosoma japonicum were detected by this Dot-ELISA, 41 samples were positive, the sensitivity was 97.6%, and all of the ten healthy rabbits' sera were negative, the specificity was 100%.All the six McAbs could recognize natural signal protein 14-3-3. It has been proved preliminarily that the Dot-ELISA based on 3F1 and 5D1 is valuable for the diagnosis of active infection of Schistosoma japonicum and accessing the chemotherapeutic effect of schistosomiasis.

Background Schistosomiasis remains an important public health problem throughout tropical and subtropical countries. Humans are infected through contact with water contaminated with schistosome cercariae. Therefore, issuing early warnings on the risk of infection is an important preventive measure against schistosomiasis. Sentinel mice are used to monitor water body infestations, and identifying appropriate antibody responses to schistosome antigens for early detection of infection would help to improve the efficiency of this system. In this study we explored the potential of detecting antibodies to the hydrophilic domain (HD) of the 23-kDa membrane protein (Sj23HD) and soluble egg antigen (SEA) of Schistosome japonicum for early detection of schistosome infection in sentinel mice. Results Development of IgM and IgG antibody levels against Sj23HD and SEA in S. japonicum infected mice was evaluated over the course of 42 days post-infection by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The Sj23HD and SEA specific IgM and IgG levels in mice all increased gradually over the course of infection, but IgM and IgG antibodies against Sj23HD presented earlier than those against SEA. Furthermore, the rates of positive antibody responses against Sj23HD were higher than those against SEA in the early stage of schistosome infection, suggesting that the likelihood of detecting early infection using anti-Sj23HD responses would be higher than that with anti-SEA responses. The use of immunoblotting could further improve the early detection of schistosome infection due to its greater sensitivity and specificity compared to ELISA. Additionally, the levels of Sj23HD and SEA specific antibodies positively correlated with the load of cercariae challenge and the duration of schistosome infection. Conclusions This study demonstrated that antibody responses to the Sj23HD antigen could be monitored for early detection of schistosome infection in mice, especially by immunoblotting which demonstrated greater sensitivity and specificity than ELISA for detection Sj23HD antibodies.

Background Schistosomiasis remains a major public health concern that afflicts millions of people worldwide. Low levels of Schistosoma infection require more sensitive diagnostic methods. In this study, a time-resolved fluoroimmunoassay (TRFIA) was developed for detecting the signal transduction protein 14-3-3, a circulating antigen of Schistosoma japonicum. Results The detection limit of 14-3-3-TRFIA was 0.78 ng/ml, with a linear measurement range from 0.78 to 800 ng/ml. The average intra-assay and inter-assay variability of this TRFIA was 8.9% and 12.2% respectively, and the mean recovery rate ranged from 92.1% to 115.5%. Within the first 21 days post-infection in rabbits, the positive rates of the 14-3-3-TRFIA were distinctly higher compared to ELISA. All these findings illustrate that 14-3-3-TRFIA has a higher detection efficacy and is a good early diagnostic method for active Schistosoma infection. Conclusions A sandwich TRFIA for detecting the circulating antigen 14-3-3 of S. japonicum has been developed, and has demonstrated to be a good potential diagnostic method for schistosomiasis.

To synthesize and express the gene of egg protein IPSE (IL-4-inducing principle of Schistosoma mansoni eggs) and evaluate its immunologic characteristics.The IPSE gene of S. mansoni was synthesized by overlapping PCR, and confirmed by DNA sequencing, The recombinant plasmid IPSE-pET32a(+) was constructed by inserting the gene of IPSE into expression vector pET32a(+) at the downstream of thioredoxin (Trx) coding sequence. The recombinant plasmid IPSE-pET32a(+) was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by IPTG. Large-scale fusion protein was prepared and purified with Ni affinity chromatography gel under denaturing conditions. A small amount of soluble Trx-IPSE was obtained by dialyzing the fusion protein in a large volume of PBS. Western blotting was used to detect if the recombinant IPSE was recognized by the IgG antibody in the pooled patient sera of schistosomiasis japonica and its binding capacity to the non-specific IgE antibody in the sera of healthy persons.DNA sequencing confirmed that the nucleotide sequence of synthesized IPSE gene was completely identical to the native one. SDS-PAGE showed that the recombinant plasmid IPSE/pET32a(+) expressed a fusion protein with an Mr 35700 after being induced by IPTG. The pure fusion protein Trx-IPSE reacted positively with the pooled sera of schistosomiasis patients under either denaturing or renaturing conditions. The protein Trx-IPSE also reacted with the nonspecific IgE in the sera of healthy persons.The IPSE gene of Schistosoma mansoni has been synthesized, and the recombinant can react with natural antibody IgG against S. japonicum and non-specifically bind to IgE antibody.

To synthesize and express the gene of TSP2 hydrophilic domain of Schistosoma japonicum, and investigate the immunogenicity of the recombinant TSP2HD protein.The whole DNA fragment encoding the TSP2 hydrophilic domain was synthesized by overlapping PCR, and confirmed by DNA sequencing. The recombinant plasmid TSP2HD-PG was constructed by inserting the purified TSP2HD DNA fragment into expression vector pGEX-4T-3 and the GST-TSP2HD fusion protein was expressed by transforming the recombinant plasmid TSP2HD-PG into Escherichia coli BL21 (DE3) and induced the recombinant with isopropyl beta-D-1-thiogalactopyranoside (IPTG). The expressing situation of fusion protein was analyzed by SDS-PAGE. The GST-TSP2HD fusion protein was purified by affinity chromatography with glutathione sepharose 4B gel, and the purified recombinant TSP2HD protein was prepared by digesting the GST-TSP2HD fusion protein with thrombin. The immuno-response of the recombinant TSP2HD recognized by the pool sera of schistosomiasis patients and the pool sera of heavily infected rabbits was explored by Western blotting analysis. The immunogenicity of the recombinant TSP2HD was investigated by comparing the difference of counts per minute (cpm) value of lymphocyte proliferation test between experiment group and control group.A 228 bp of TSP2HD gene fragment was obtained after overlapping PCR of three times and its DNA sequence was confirmed by DNA sequencing, which was same to one of the native TSP2HD. The recombinant containing recombinant plasmid TSP2HD-PG expressed a soluble fusion protein of GST-TSP2HD (Mr approximately 34 000) after being induced with IPTG. The purified recombinant TSP2HD protein was obtained through digesting the GST-TSP2HD fusion protein with thrombin. The recombinant TSP2HD was recognized by pool sera of schistosomiasis patients and pool sera of infected rabbits, indicating that the recombinant TSP2HD has a good response activity. The recombinant TSP2HD also stimulated proliferation of lymphocytes in infected mouse, the cpm value of experiment group was higher than that of the control (P0.01).The Sj TSP2HD gene has been synthesized and expressed with immunogenicity which is similar to that of the native antigen.

Experimental vaccination with radiation-attenuated cercariae (RAC) confers possible practical levels of resistance to challenge infection by humoral and by cellular mechanism. Here, we aimed to identify possible vaccine antigens by using specific IgG antibody from RAC vaccinated miniature pig. Two milligrams of soluble egg antigen (SEA) or schistosomal worm antigen preparation (SWAP) was fractionated using two dimensional liquid chromatography (proteome PF 2D) consisted of high performance chromatofocusing (HPCF) and high resolution reversed phase chromatography (HPRP). Of the 42 HPCF fractions of SEA or SWAP, 26 (61.9%) or 15 (35.7%) showed positive dot blot reaction with RAC vaccinated serum respectively. The dot blot positive fractions were applied to the second HPRP column. One hundred and seven out of 26 x 96 of SEA fractions and 18 out of 15 x 96 SWAP fractions reacted with RAC vaccinated serum. From the positive fractions we chose 17 of SEA and 10 of SWAP that had no reactivity with normal cercariae infected (NCI) sera and had single peak of 214 nm; and automated N-terminal amino acid sequence based on in situ Edman Reaction was conducted. Four sequences were obtained and applied to the homology search in NCBI database. A total of eight candidate genes were listed up and their cDNA clones from schistosomula stage were obtained. Two of the recombinant proteins (AAW27472.1 and AXX25883.1) showed strong reactivity with the RAC vaccinated serum but marginal with NCI serum. This protocol using proteome PF 2D could be applicable in identifying immunoreactive proteins from crude extract for the development of vaccines or for diagnostics., Parasitology International, 58(1), pp.36-44; 2009

The granulomatous reaction which occurs around egg trapped in the intrahepatic venules ultimately may lead to fibrosis, which is the main pathogenesis of schistosomiasis. The excreted proteins from eggs play an important role during this process, and they may be a target for developing new strategies to control the hepatic pathogenesis caused by schistosome infection. In this study, fifteen genes encoding secreted or membrane binding protein were identified with the signal sequence trapping method by retrovirus mediated expression screening (SST-REX) of cDNAs from the egg of Schistosoma japonicum (Chinese strain).

To express the miracidial antigen from eggs of Schistosoma japonicum (Chinese mainland strain) (SjMP10), and investigate the role of the miracidial antigen during the hepatic granuloma formation of schistosomiasis.A pair of specific primers was designed and synthesized according to the nucleotide sequence of the open reading frame of the miracidial antigen gene. The open reading frame of the miracidial antigen gene was amplified, digested by restrictive enzyme (BamHI, SolI), and cloned directly into the expression plasmid pGEX-4T-3 to construct the recombinant plasmid. The recombinant plasmids were transformed into E. coli BL21(DE3), and induced by IPTG to express the fusion protein of GST-SjMP10. The expressed fusion protein was purified by electric elution method, and its antigenicity was examined by Western blotting and lymphocyte proliferation test.The gene of miracidial antigen was cloned into the expression plasmid pGEX-4T-3. After induced by IPTG, the recombinant expressed a fusion protein of GST-SjMP10, with a molecular weight of 39 000 approximately. The purified fusion protein showed proper antigenicity that could be recognized by the sera of rabbits heavily infected by Schistosoma japonicum and could stimulate the proliferation of splenic lymphocytes of infected BALB/c mice.The miracidial antigen from eggs of Schistosoma japonicum was expressed successfully.

To study the expression of nitric oxide synthase (NOS) mRNA in the gonad of Oncomelania hupensis in different temperature.The snails were cultured at temperature of 0 degree C, 15 degrees C and 25 degrees C for 1 month. The total RNA of each group was extracted with the RNA extraction kit. A pair of degenerate primers was designed from conserved regions of mammalian NOSs, and the expression of NOS mRNA in the gonad was measured by means of reverse transcription polymerase chain reaction (RT-PCR).The target genes of NOS were detected in the gonad of snails. The level of expression of snail NOS mRNA in 25 degrees C group and 0 degree C group was significantly higher than that in the control group(P0.01), there was no significant difference for the expression products between 15 degrees C group and control group (P0.05).The designation of primers of the snails was validated. The impact of temperature on the expression of snail NOS mRNA was determined, which suggested that NO plays an important role in the physiological and pathological modulation.

To observe the protective immunity induced by the nucleic acid vaccine of 21.7 kDa membrane protein molecule of Schistosoma japonicum Chinese mainland strain (SjC 21.7) in BALB/c mice.A pair of primers (P1 and P2) was synthesized according to the DNA sequence of the SjC21.7. The ORF sequence of SjC21.7 was amplified by PCR, and the Kozark sequence was added to the position of initiator. The gene fragment was inserted into the eukaryotic expression plasmid pcDNA3.1 to form the recombinant plasmid SjC21.7-pcDNA3.1. Forty-eight BALB/c mice were divided into three groups: control, test and boost. Each mouse was injected in quadriceps femoris with plasmid pcDNA3.1 (control) or recombinant plasmid SjC21.7-pcDNA3.1 (test, boost); for the boost group, with additional P35-pcDNA3.1 and P40-pcDNA3.1. All mice were immunized three times with an interval of 2 weeks, challenged each with 45 cercariae of S. japonicum at the 30th day after final immunization. At day 45 after challenge, all mice were sacrificed, the numbers of worms and hepatic eggs were counted. Antibody level in the sera of mice before and two weeks after immunization was determined with ELISA. The expression of the target gene in quadriceps femoris was observed with immunohistochemistry.The immunohistochemistry analysis showed that there were specific antigens expressed in the local tissue of the test group mice. There was specific IgG in the serum of partial mice in test and boost groups. Compared with the control group, the worm reduction rate was 29.9% and its egg reduction rate 13.8% in the test group; 31.9% and 28.0% respectively in the boost group. The egg reduction rate in the boost group was higher than that of the test group (P0.05).The SjC21.7 nucleic acid vaccine could induce partial protective immunity against Schistosoma japonicum in BALB/c mice.

Background We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. Methodology/Principal Findings Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin)-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10−6 M) and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. Conclusions The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation), and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted., Author Summary While isolating membrane-bound and secreted proteins as targets for Schistosoma japonicum vaccine, we identified a novel potentially functional gene family which had originated by a gene duplication mechanism. Here, we integrated structural homology modeling and biochemical methods to show that this gene family encodes proteins with sea-urchin sperm protein, enterokinase and agrin (SEA) –domain, with heme-binding properties. Typical of SEA-structural domains, the characterized proteins specifically interacted with glycosaminoglycans (GAGs), with implication in ligand gathering and immune-evasion. Consistent with modeled heme-binding pocket, we observed high affinity heme-binding and spectroscopic attributes of hexa-coordinated heme iron. Localization of the native gene-products on adult worm tegument and gastrodermis, host interfaces for heme-sequestration and acquisition, suggests potential roles for this gene family in heme-detoxification and heme-iron uptake.

Background Schistosome infection typically induces a polarized Th2 type host immune response. As egg antigen molecules play key roles in this immunoregulatory process, clarifying their functions in schistosomiasis would facilitate the development of vaccine and immunotherapeutic methods. Schistosoma japonicum (Sj) CP1412 (GenBank: AY57074.1) has been identified as a new member of the RNase T2 family with immune regulatory functions. Methods The expression plasmid Sj CP1412-pET28a was constructed and transformed into bacteria for production of recombinant Sj CP1412 protein (rSj CP1412) via IPTG induction. The RNase activity of Sj CP1412 was predicted by bioinformatic analysis and confirmed by digesting the yeast tRNA with rSj CP1412.C57BL/6j mice were immunized with rSj CP1412, and its immune regulatory effects in vivo and in vitro were investigated. Meanwhile, the relationship between the RNase activity of Sj CP1412 and its immune regulation was observed. Results Sj CP1412 was confirmed as a novel RNase T2 family protein with RNase activity. Immunoblotting and RT-PCR analyses demonstrated Sj CP1412 as a protein exclusively secreted/excreted from eggs, but not cercariae and adult worms. Stimulating RAW264.7 macrophages with rSj CP1412 raised the expression of CD206, Arg-1 and IL-10, which are related to M2 type macrophage differentiation. Stimulating dendritic cells (DCs) with rSjCP1412 failed to induce their maturation, and the recombinant protein also inhibited LPS-stimulated DC maturation. Depletion of Sj CP1412 from soluble egg antigen (SEA) impaired the ability of SEA to induce M2 type polarization of RAW264.7 macrophages. Immunizing mice with rSj CP1412 induced high antibody titers, increased serum IL-4 and TGF-β levels and splenic CD4 + CD25 + Foxp3 + T cells, downregulated serum IFN-γ levels and alleviated the egg granuloma pathology of schistosome infection. In vitro stimulation by rSj CP1412 significantly increased CD4 + CD25 + Foxp3 + T cell numbers in splenocytes of healthy mice. The rSj CP1412 protein with RNase activity inactivated by DEPC failed to induce M2 surface marker CD206 expression in RAW264.7 macrophages. Conclusions The Sj CP1412 protein expressed specifically in S. japonicum eggs is a novel member of the RNase T2 family. Similar to Omega-1 of Schistosoma mansoni, the Sj CP1412 protein drives polarization of the host Th2 immune response, which is dependent on its RNase activity. These data provide new evidence towards understanding the immune regulatory role of RNase T2 family proteins during schistosome infection. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1962-y) contains supplementary material, which is available to authorized users.