SjHSP90 recombinant protein and its possible application in the diagnosis of schistosomiasis and treatment efficacy monitoring.

PURPOSE: The fraction antigen of Schistosoma japonicum (107-121 kDa) eggs can be used for treatment efficacy monitoring, but the methods are laborious. This study analyzed the antigen and its feasibility for infection screening and treatment efficacy monitoring, which is the key to schistosomiasis control. METHODS: The fraction antigens have been analyzed by shotgun mass spectrometry. The recombinant proteins of candidates from the fraction antigens have been prokaryotic expression and purification in large amounts with high purity. The sera have been collected from rabbits and mice models of schistosomiasis infection and treatment. ELISA evaluated the diagnostic value of the candidate proteins. RESULTS: SJCHGC00820 and SJCHGC06900, with higher credibility, were identified through Shotgun mass spectrometry. ELISA results showed that rSj00820 has a diagnostic value for schistosomiasis (positive OD/negative OD P/N=3.6), while rSj06900 showed negative (P/N)<2. In rabbits, the specific serum antibodies for SjHSP90(rSj00820) in the infected animals peaked 6 weeks after infection and gradually decreased after treatment, reaching negative levels at 11 weeks. SjHSP90-ELISA was used to test serum samples from infected mice. The sensitivity and specificity reached >90%, similar to the diagnostic value obtained with soluble egg antigen (SEA) (SEA-ELISA). After treatment, the negative conversion rate reached >80%, significantly superior to SEA-ELISA. CONCLUSIONS: The SjHSP90-ELISA can be used for the immunological diagnosis and treatment efficacy monitoring of schistosomiasis. The study lays a foundation for further developing screening and diagnostic kits.

Electrogenerated chemiluminescence biosensor for detection of mercury (II) ion via target-triggered manipulation of DNA three-way junctions.

A new electrogenerated chemiluminescence (ECL) biosensor is fabricated for the determination of mental ion incorporating DNA three-way junction structure (DNA-TWJ). As a model system, Hg2+ was chosen as an analyte. The ECL biosensor was fabricated by covalently coupling Hg2+ special DNA-TWJ tagged with ruthenium (II) complex (Ru) (named TW/Ru-J1) to the surface of glassy carbon electrode that had been covalently modified with 4-aminobenzoic acid via electrochemical oxidations. Upon binding of Hg2+ to the TW/Ru-J1, the confirmation of TW/Ru-J1 changed and induced Ru away from surface of electrode and thus led to a low ECL signal. The signal linearly decreases with the concentration of Hg2+ in the range from 0.1 pM to 10 pM with a detection limit of 0.04 pM This study could be easily extended to various analytical platforms for the detection of many kinds of analytes or their interactions such as DNA/RNA, DNAzyme/target, aptamer/target, and antibody/antigen.

Labeled and non-label electrochemical peptide inhibitor-based biosensing platform for determination of hemopexin domain of matrix metalloproteinase-14.

Two labeled and non-label electrochemical peptide-based biosensors for highly sensitive and selective determination of hemopexin domain of matrix metalloproteinase-14 (PEX-14) were reported for the first time. Herein, the thiolated PEX-14 binding peptide inhibitor (ISC) or the ISC-tagged with ferrocene carboxylic acid (CIS-Fc) was self-assembled on the surface of gold electrode tested for binding with PEX-14 by non-label electrochemical impedance spectroscopy or labeled electrochemical (L-EC) transducers. The two biosensors were compared for the analytical performance of the detection of PEX-14 with detection limit of 0.3 pg mL-1 (for L-EC biosensor) and 0.03 ng mL-1 (for EIS biosensor). This work demonstrates that probing the PEX of MMPs represents a novel approach to assess MMPs-mediated cancer dissemination and provide a platform for screening the inhibitors of Matrix metalloproteinases.

Enhancement of protective efficacy through adenoviral vectored vaccine priming and protein boosting strategy encoding triosephosphate isomerase (SjTPI) against Schistosoma japonicum in mice.

BACKGROUND: Schistosomiasis japonica is a zoonotic parasitic disease; developing transmission blocking veterinary vaccines are urgently needed for the prevention and control of schistosomiasis in China. Heterologous prime-boost strategy, a novel vaccination approach, is more effective in enhancing vaccine efficacy against multiple pathogens. In the present study, we established a novel heterologous prime-boost vaccination strategy, the rAdV-SjTPI.opt intramuscular priming and rSjTPI subcutaneous boosting strategy, and evaluated its protective efficacy against Schistosoma japonicum in mice. METHODOLOGY/PRINCIPAL FINDINGS: Adenoviral vectored vaccine (rAdV-SjTPI.opt) and recombinant protein vaccine (rSjTPI) were prepared and used in different combinations as vaccines in a mouse model. The specific immune responses and protective efficacies were evaluated. Furthermore, the longevity of protective efficacy was also determined. Results showed that the rAdV-SjTPI.opt priming-rSjTPI boosting strategy elicited higher levels of specific IgG responses and broad-spectrum specific cellular immune responses. The protective efficacy could reach up to nearly 70% and 50% of protection could be observed at 10 weeks after the last immunization in mice. CONCLUSIONS/SIGNIFICANCE: The rAdV-SjTPI.opt intramuscular priming-rSjTPI subcutaneous boosting vaccination strategy is a novel, highly efficient, and stable approach to developing vaccines against Schistosoma japonicum infections in China.

PAMAM-Lys, a novel vaccine delivery vector, enhances the protective effects of the SjC23 DNA vaccine against Schistosoma japonicum infection.

BACKGROUND: Schistosomiasis japonica remains a major public-health concern in China. Praziquantel-based chemotherapy effectively reduces both infections and intensity; however, it can not prevent re-infection. Furthermore, there is an increasing concern about praziquantel resistance following long-term repeated use of the drug in endemic areas. Therefore, development of a schistosomiasis vaccine, as a strategy to prevent and control schistosomiasis japonica, has been given high priority. The present study was conducted to develop PAMAM dendrimers as a novel vaccine delivery vector for a schistosomiasis japonica DNA vaccine and evaluate its ability to enhance protective effects against Schistosoma japonicum infection. METHODOLOGY/PRINCIPAL FINDINGS: Lysine was used to modify 4.0G PAMAM, and the modified product PAMAM-Lys was synthesized. PAMAM-Lys showed both high transfection and low cytotocity for gene delivery in vitro. DNA vaccines combined with PAMAM-Lys produced higher level of protection compare with naked DNA vaccines against S. japonicum infection in a mouse model. Futhermore,antibodies from mice immunized with PAMAM-Lys combined DNA vaccines were significantly higher than those of mice immunized with the naked DNA vaccines. The PAMAM-Lys vector elicited a predominantly IgG2a antibody response and a tremendously increase in the production of IL-2 and IFN-γ. CONCLUSION/SIGNIFICANCE: Lysine-modified PAMAM-Lys is an excellent vector. PAMAM-Lys may enhance the immunoreactivity of DNA vaccine and increase the protective effect of the SjC23 DNA vaccine against S. japonicum infection.

Construction and evaluation of replication-defective recombinant optimized triosephosphate isomerase adenoviral vaccination in Schistosoma japonicum challenged mice.

Schistosomiasis is an endemic, zoonotic parasitic disease that remains a public health concern in China. Development of transmission blocking veterinary vaccines against Schistosoma japonicum infection is urgently needed. Replication-defective adenoviral vector is an efficient vaccine delivery system that has been widely used. Its use is associated with high levels of gene insertion and expression. It is easy to construct and prepare, and is safe. It is not known whether this delivery system can improve the protective effect of schistosome vaccination. Triosephosphate isomerase from S. japonicum (SjTPI) is a promising vaccine candidate. Thus far it has induced only partial protection in animal models and needs to be further enhanced to be effective. We constructed a replication-defective adenoviral vector-based vaccine with optimized SjTPI (rAdV-SjTPI.opt). The specific immune responses and protective efficiency in mice were evaluated. Results showed that intramuscular rAdV-SjTPI.opt induced Th1 biased immune responses in the host, while subcutaneous rAdV-SjTPI.opt induced Th2 predominant immune responses. Oral rAdV-SjTPI.opt induced low levels of immune responses and no significant protection. Intramuscular rAdV-SjTPI.opt provided a consistent and repeatable higher protective effect in mice (more than 50%). These findings may be due to the associated higher levels of specific Th1, antibody responses and partially lower level of IL-17A. This report provides a foundation for developing transmission-blocking veterinary vaccines in larger animals.

A novel immunodiagnostic assay to detect serum antibody response against selected soluble egg antigen fractions from Schistosoma japonicum.

BACKGROUND: Schistosomiasis japonica remains a real threat to public health in China. The currently used immunodiagnostic assays are sensitive and have a certain degree of specificity, however, they all use complex crude antigens, are based on detection of schistosome-specific antibodies, and have been shown to cross-react with other parasitic diseases. Therefore, these assays cannot be used to evaluate chemotherapy efficacy. The development of highly sensitive and highly specific immunodiagnostic techniques that can monitor the decline of antibodies specific for S. japonica will be extremely valuable as part of the ongoing strategy to control schistosomiasis in endemic areas. Here we report on the identification of unique fraction antigens of soluble egg antigen (SEA) to which the antibodies disappear 7 weeks after effective treatment. Furthermore, we use these SEA fractions to develop a modified assay with both high sensitivity and specificity. METHODOLOGY/PRINCIPAL FINDINGS: SEA of S. japonicum was fractionated by electrophoresis using 7.5% SDS-PAGE under non-reducing conditions. The SEA fraction antigens to which antibodies were decreased soon after treatment were collected and used as the detection antigens to establish the FA-ELISA. Sera from patients with acute and chronic schistosomiasis infection, healthy people, and those with other parasitic diseases, were used to evaluate their sensitivity and specificity. Furthermore, sera from patients with chronic schistosomiasis infection were evaluated before and after treatment at different time points to evaluate their chemotherapeutic efficacy. CONCLUSION/SIGNIFICANCE: We demonstrated that this novel FA-ELISA provided high sensitivity and specificity, with very low cross-reactivity, and can serve as an effective tool to determine the efficacy of chemotherapy against S. japonicum.

Positive diversifying selection on Plasmodium vivax RON2 protein.

Plasmodium rhoptry neck protein 2 (RON2), which is released from the neck portion of the merozoite rhoptries and interacts with the microneme protein Apical Membrane Antigen 1 (AMA1), plays a crucial role in erythrocyte invasion. In this study, we sequenced the Plasmodium vivax RON2 gene from 19 P. vivax isolates collected in central China in order to establish whether this protein is under positive diversifying selection, which may occur as a result of protective host immune pressure†. In comparison with the P. vivax Sal-1 reference line, we found 10 amino acid substitutions dispersed throughout the open reading frame as well as indels caused by polymorphism in a repeat unit (21-23 repeats of (Q/E/K/N/H)(G/D)G(H/L/Y/P)G) in the second tandem repeat region located at amino acid positions 541-650. A McDonald-Kreitman test with RON2 sequences from the primate malaria parasite Plasmodium knowlesi, detected significant departure from neutrality in the PvRON2 3' region (nucleotide positions 2668-6609). These results suggest that the PvRON2 gene has evolved under positive diversifying selection.

[Anti-atherogenic effect and its mechanisms of soluble egg antigen of Schistosomia japonicum in ApoE-/- mice].

OBJECTIVE: To study the preventive effects of soluble egg antigen (SEA) of Schistosomia japonicum on atherosclerosis in ApoE-/- mice and its immune modulatory mechanisms. METHODS: ApoE-/- mice were divided into an Experimental Group One and an Experimental Group Two. The mice in the Experimental Group One dividing into a prevention and a control subgroups were fed with high fat diet since the first week, the mice in the former subgroup were injected intraperitoneally with SEA while those in the latter one were injected with phosphate buffered saline (PBS) with 1 week interval for 4 times. The mice in the Experimental Group Two were fed with high fat diet for 14 weeks, and then they were divided into a treatment and a control subgroups, which were injected with SEA and PBS, respectively, since the 14th week with 1 week interval for 4 times. All the mice were killed in the 22nd week, and the atherosclerosis development and the change of levels of cytokines and CD4+CD25+ FoxP3+T cells in mice were observed. RESULTS: Immunization with SEA led to a significant reduction in the levels of cholesterol, TNF-alpha, and IL-10 in all the ApoE-/- mice. The atherosclerosis plaque area of aorta of ApoE-/- mice in the prevention subgroup reduced obviously, while there was no significant change in the treatment subgroup. In the 22nd week, the proportion of CD4+CD25+ FoxP3+T cells population in CD4+ T cells was (4.4 +/- 0.9)% in the prevention subgroup and there was a significant difference compared with that of the control subgroup [(2.6 +/- 0.3)%] (P < 0.05). However, the change of the proportion in the treatment subgroup showed no statistic significance (P > 0.05). CONCLUSION: The preventive injection of SEA in ApoE-/- module mice has the effect of anti-atherosclerosis by increasing the proportion of Treg cells together with the inhibition of inflammatory cytokines at the beginning of disease.

Construction and characterization of an anti-asialoglycoprotein receptor single-chain variable-fragment-targeted melittin.

Antibody-therapeutic agent conjugation to be delivered specifically to tumor cells is required for many target-based therapeutic strategies. In the present study, a recombinant immunotoxin was constructed by which melittin was fused to an anti-asialoglycoprotein receptor (ASGPR) single-chain variable fragment antibody (C1), and targeting ability and cytolytic efficacy of the fusion protein were studied. Our results suggested that the recombinant 29.4 kDa protein C1M was expressed in Escherichia coli as a soluble style. Binding of C1M to the surface of hepatocellular carcinoma (HCC) cells was confirmed by both immunohistochemistry and flow cytometry assays. C1M kept the hemolytic activity of melittin and exhibited cytolytic capacity to HepG2 cells at a concentration of 1.5 µg/mL, under which erythrocytes would not be lysed. The effects were greatly inhibited by coadministration with asialoorosomucoid, a natural ligand for ASGPR. These results suggested that C1M conferred targeting and ASGPR-specific cytotoxicity to HCC cells. This work makes it possible to further investigate its antihepatoma efficacy in vivo.

Oral delivery of the Sj23LHD-GST antigen by Salmonella typhimurium type III secretion system protects against Schistosoma japonicum infection in mice.

BACKGROUND: Schistosomiasis japonica is a zoonotic parasitic disease and oral vaccine delivery system would be benefit for prevention of this disease. Although attenuated salmonella has been used as an antigen expression vector for oral vaccine development, the membrane-bound vacuoles in which bacteria reside hinders the presentation of expressed heterologous antigens to the major histocompatibility complex (MHC) molecules. The present work used an attenuated Salmonella typhimurium strain VNP20009 to secretory expression of Sj23LHDGST bivalent antigen from Schistosoma japonicum and tested the protective efficacy against S. japonicum infection in orally immunized mice. METHODOLOGY/PRINCIPAL FINDINGS: Promoters (nirB or pagC) were used to express the antigen (Sj23LHDGST) and the Salmonella type III or α-hemolysin secretion system was employed to secrete it. The immunoblotting analysis and fluorescent microscopy revealed that the antigen was effectively expressed and delivered to the cytosol of macrophages in vitro. Among recombinant vaccine strains, an engineered VNP20009 which expressed the antigen by nirB promoter and secreted it through type III secretion system (nirB-sopE(1-104)-Sj23LHD-GST) efficiently protected against S. japonicum infection in a mouse model. This strain elicited a predominantly IgG(2a) antibody response and a markedly increase in the production of IL-12 and IFN-γ. The flow cytometric analysis demonstrated that this strain caused T cell activation as evidenced by significantly increased expression of CD44 and CD69. CONCLUSION/SIGNIFICANCE: Oral delivery of antigen by nirB-driven Salmonella typhimurium type III secretion system is a novel, safe, inexpensive, efficient and convenient approach for schistosome vaccine development.

Expression, characterization and therapeutic efficacy of chimeric Fab of anti-idiotypic antibody NP30 against Schistosoma japonicum.

The murine monoclonal anti-idiotypic antibody NP30 is a promising therapeutic antibody against Schistosoma japonicum. However, the immunogenicity of murine NP30 limits its further study and application in humans. Here the chimeric Fab of NP30 (chFab-NP30) comprising the variable regions of murine NP30 and constant regions of human antibody was assembled. chFab-NP30 was expressed and purified as a soluble and functional protein. Administration of chFab-NP30 in vivo increased the survival rate, reduced egg burdens and ameliorated organ pathology of mice with acute schistosomiasis. Our study indicated that chFab-NP30 is a promising candidate to be used as a specific and efficient recombinant antibody against acute schistosomiasis japonica. Further studies on function mechanism of chFab-NP30 needs to be carried out in the future.

The efficacy of NP11-4-derived immunotoxin scFv-artesunate in reducing hepatic fibrosis induced by Schistosoma japonicum in mice.

Schistosomiasis is one of the most prevalent parasitic diseases in China, and hepatic fibrosis caused by schistosome infection is the principal cause of death. The aim of this study was to evaluate the efficacy of NP11-4-derived immunotoxin scFv-artesunate on Schistosoma japonicum-induced hepatic fibrosis. A single-chain variable fragment (scFv) was generated from the murine anti-Schistosoma japonicum (S. japanicum) monoclonal antibody NP11-4. The scFv was expressed as a soluble protein and purified by Ni-affinity chromatography. After conjugation with artesunate, the binding ability with soluble egg antigens (SEA) was determined by an enzyme-linked immunosorbent assay (ELISA). The biological activity of purified scFv, scFv-artesunate (immunotoxin), and artesunate was detected in vivo. Image-Pro Plus software was used to analyze the size of egg granuloma and the extent of liver fibrosis. The recombinant scFv expession vector was constructed and expressed successfully. After purification by a His-trap Ni-affinity column, the scFv yield was approximately 0.8 mg/L of culture medium. ELISA results showed that chemical conjugation did not affect the binding activity of the immunotoxin. Our animal experiments indicated that the immunotoxin could significantly reduce the size of egg granuloma in the liver and inhibit hepatic fibrosis. The immunotoxin could be used as a promising candidate in the targeted therapy of S. japonicum-induced hepatic fibrosis.

Synergistic enhancement of immunogenicity and protection in mice against Schistosoma japonicum with codon optimization and electroporation delivery of SjTPI DNA vaccines.

Schistosomiasis is an endemic, zoonotic parasitic disease caused by Schistosoma japonicum that remains a public health concern and an effective vaccine is needed. Triose-phosphate isomerase from S. japonicum is a promising schistosome vaccine antigen shown to be immunogenic when delivered as a DNA vaccine, however, the previous S. japonicum triose-phosphate isomerase (SjTPI) DNA vaccine needs to be further optimized to achieve higher protection. In the current study, codon optimization of SjTPI DNA insert, combined with electroporation but not with the addition of a tPA leader or heat-shock protein in-frame with the SjTPI gene insert, enhanced Th1-type antibody and cytokine production and most significantly, achieved great than 50% reduction of infection against challenge with S. japonicum cercariae, a major milestone in S. japonicum vaccine development. Our results suggest that the combination of a codon optimized vaccine design and an efficient vaccine delivery system can greatly improve the potential of a SjTPI DNA vaccine as a viable schistosome vaccine candidate.

DNA vaccination by electroporation and boosting with recombinant proteins enhances the efficacy of DNA vaccines for Schistosomiasis japonica.

Schistosomiasis japonica is an endemic, zoonotic disease of major public health importance in China. Control programs combining chemotherapy and snail killing have not been able to block transmission of infection in lakes and marsh regions. Vaccination is needed as a complementary approach to the ongoing control programs. In the present study, we wanted to determine if the efficacies of DNA vaccines encoding the 23-kDa tetraspanin membrane protein (SjC23), triose phosphate isomerase (SjCTPI), and sixfold-repeated genes of the complementarity determining region 3 (CDR3) in the H chain of NP30 could be enhanced by boosting via electroporation in vivo and/or with cocktail protein vaccines. Mice vaccinated with cocktail DNA vaccines showed a significant worm reduction of 32.88% (P < 0.01) and egg reduction of 36.20% (P < 0.01). Vaccine efficacy was enhanced when animals were boosted with cocktail protein vaccines; adult worm and liver egg burdens were reduced 45.35% and 48.54%, respectively. Nearly identical results were obtained in mice boosted by electroporation in vivo, with adult worm and egg burdens reduced by 45.00% and 50.88%, respectively. The addition of a protein vaccine boost to this regimen further elevated efficacy to approximately 60% for adult worm burden and greater than 60% for liver egg reduction. The levels of interleukin-2, gamma interferon, and the ratios of immunoglobulin G2a (IgG2a)/IgG1 clearly showed that cocktail DNA vaccines induced CD4(+) Th1-type responses. Boosting via either electroporation or with recombinant proteins significantly increased associated immune responses over those seen in mice vaccinated solely with DNA vaccines. Thus, schistosome DNA vaccine efficacy was significantly enhanced via boosting by electroporation in vivo and/or cocktail protein vaccines.

[Enhancing protective immunity effects of TPI DNA vaccine against Schistosoma japonicum through codon optimization].

OBJECTIVE: To study the protective effect of codon optimized TPI DNA vaccine against Schistosoma japonicum infection. METHODS: Sixty female BALB/c mice were randomly divided into 5 groups. The mice were injected through musculus quadriceps femoris with 100 microg pcDNA 3.1 control (Group A), pcDNA3.1-TPI (Group B), pcDNA 3.1-TPI-mHSP70 (Group C), pcDNA3.1-TPI.opt (Group D), and pcDNA3.1-TPI.opt-mHSP70 (Group E) respectively. All mice were immunized for three times with an interval of two weeks. The mice were challenged with (40+/-1) cercariae of S. japonicum per mouse by abdominal skin penetration 4 weeks after the last immunization, and sacrificed at 42 days post-challenge, the number of worms or hepatic eggs was counted. Blood was taken for the detection of IgG, IgG1, and IgG2a 2 days before immunization and before challenge, respectively. Spleen cells of 2 mice from each group were cultured and stimulated with ConA and rSjCTPI peptide, and the supernatant was collected for detection of IL-2, IL-4, IL-5, IFN-gamma, and TNF by flow cytometry. RESULTS: ELISA showed that the mice in groups B, C, D, and E produced specific IgG and IgG1, IgG2a antibody isotypes, and the ratio of IgG2a/IgG1 was 1.73, 2.06, 2.44, and 3.09, respectively. The levels of IL-2, IFN-gamma and TNF in groups D and E were higher than that of groups B and C. The worm reduction rate and hepatic egg reduction rate in groups D (36.03%, 41.7%) and E (39.03%, 46.85%) were higher than those of groups B (26.28%, 28.35%) and C (28.38%, 31.39%) (P<0.01) . CONCLUSIONS: The codon optimized TPI DNA vaccine induces higher level of protective effect and Th1-biased cellular immune response than those of non-optimized TPI DNA vaccine.

A novel molluscicidal formulation of niclosamide.

Snail control by molluscicides is an important strategy for schistosomiasis control in China. Currently, only one chemical molluscicide, niclosamide, which is used as 50% wettable powder of niclosamide ethanolamine salt (WPN), is commercially available for field snail control in China. However, WPN is costly, toxic, and has a lower dispersibility and precipitates rapidly. In this paper, we describe the development of a novel formulation of niclosamide, a suspension concentrate of niclosamide (SCN). The efficacy of SCN was evaluated both in the laboratory and field. SCN showed better molluscicidal effects than conventional formulation of WPN, as determined by LC(50) for adult snails, young snails, and snail eggs. The acute toxicity of SCN to Brachdanio rerio hamiton was less than WPN. In conclusion, the novel formulation of SCN suspension is physically more stable, more effective, and less toxic. Therefore, it can be more useful for controlling snails in endemic areas of schistosomiasis in China.

[Development and identification of the multiple B cell epitope antigens of Schistosoma japonicum].

OBJECTIVE: To develop multiple B cell epitope antigens of Schistosoma japonicum and evaluate their antigenicity. METHODS: Bioinformatics software BioSun was used to predict B cell epitopes from Sj22.6, Sj14-3-3 and Sj26. The predicted epitopes P2, P6 and P7 were ligated to construct P2-P6-P7 and P6-P2-P7 multiepitope in random order, a 6 amino acid linker inserted between epitopes. Recombinant plasmids containing the two multiepitopes identified by enzyme digestion and sequencing were transformed into E. coli BL21. The expressed recombinant fusion proteins of E. coli BL21 induced with IPTG were purified with Ni2+ chelating HiTrap HP column. Their antigenicity was evaluated with Western-blotting. RESULT: The two multiple B cell epitopes P2-P6-P7 and P6-P2-P7 were successfully cloned into pET-32c(+) plasmid and fusion proteins were expressed. SDS-PAGE showed a single band and both of the recombinant fusion proteins were with Mr 20 400. The two proteins reacted with the sera of schistosomiasis patients but not with that of healthy people. CONCLUSION: Two multiple B cell epitope antigens were developed with potential diagnosis value.

Epidemiology of schistosomiasis in the People's Republic of China, 2004.

Results from the third nationwide cluster sampling survey on the epidemiology of schistosomiasis in the People's Republic of China, conducted by the Ministry of Health in 2004, are presented. A stratified cluster random sampling technique was used, and 239 villages were selected in 7 provinces where Schistosoma japonicum remains endemic. A total of 250,987 residents 6-65 years of age were included in the survey. Estimated prevalence rates in the provinces of Hunan, Hubei, Jiangxi, Anhui, Yunnan, Sichuan, and Jiangsu were 4.2%, 3.8%, 3.1%, 2.2%, 1.7%, 0.9%, and 0.3%, respectively. The highest prevalence rates were in the lake and marshland region (3.8%) and the lowest rates were in the plain region with waterway networks (0.06%). Extrapolation to all residents in schistosome-endemic areas indicated 726,112 infections. This indicates a reduction of 16.1% compared with a nationwide survey conducted in 1995. However, human infection rates increased by 3.9% in settings where transmission is ongoing.

[Expression and bioactivity of S1 scFv antibody against SAG1 of Toxoplasma gondii fused with green fluorescent protein].

OBJECTIVE: To construct, express and purify human scFv antibody (S1) against the recombinant SAG1 of Toxoplasma gondii and fused to green fluorescent protein (GFP), and observe its binding capacity to tachyzoite of Toxoplasma gondii. METHODS: The GFP gene amplified from vector pEGFP-N1 was subcloned into procaryotic expression vector pET-26b (+), then the S1 scFv antibody gene amplified from phagmid pIT-2-S1 was cloned into downstream of GFP gene. The recombinant plasmid pET-26b-GFPS1 proved by DNA sequencing was transformed into E. coli BL21, and induced for fusion expression of GFPS1 with IPTG, the green fluorescence of E. coli BL21 harboring plasmid pET-26b-GFPS1 was observed under the fluorescence microscope. The expressed GFPS1 was purified with Ni2+ chelating HiTrap HP column, and detected with SDS-PAGE. Toxoplasma tachyzoites were incubated with the recombinant GFPS1, and the binding bioactivity was observed under the fluorescence microscope. RESULTS: The fused gene of S1 and GFP was successfully cloned into procaryotic expression vector pET-26b proved by DNA sequencing. The green fluorescence of E. coli BL21 harboring plasmid pET-26b-GFPS1 was catched under the fluorescence microscope. The recombinant GFPS1 protein about Mr 53 000 was expressed in E. coli as inclusion body. The immunofluorescence detection verified that anti-rSAG1 scFv antibody S1 could specifically bind outer membrane of Toxoplasma tachyzoite. CONCLUSIONS: The purified rGFPS1 shows a strong binding capacity to outer membrane of Toxoplasma tachyzoite using GFP as a labeling protein, and the constructed pET-26b-GFP can also be used for research on other targeting molecules.