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This study reported the primary application of FA-ELISA from 107-121kDa fration antibody of Schistosoma Japonicum for detection of the short-term antibody in patients with schistosomiasis. The result showed that this method provided high sensitivity (with positive rate of 93. 33% with 30 cases of schistosomiasis) and good specificity (no false postive in 60 health objects). When one group of schistosome patients were examined with FA -ELISA and with the routine SEA-ELISA before chemotherapy and at 8 months,1 year and 1. 5 year after treatment,it showed good property for evaluation of chemotherapy efficacy with FA -ELISA,which was much better than the routine method.
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Objective To explore the Schistosoma japonicum cercariae collected method and the quantity of cercariae obtained from a great quantity artificial infected snails. Methods In laboratory condition, Oncomelania snails were infected with schistosome miracidia. Sixty days post-infection all snails were divided into 7 shares. Cercariae shed from 1 share snails every day and the number of all shedding days were 40. Cercariae shed from snails were collected with low-velocity centrifuge and the cercariae sediment were weighted. Results One thousand and nine hundred g snails bred for 120 days post-infection, the infection and survival rates were 36. 00% and 51. 58%. Cercariae col-lected were 10. 5 g from 40 days collection. Cercariae quantity of shedding from 1 000 positive snails per day was 0. 257 4 g.
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Objective To investigate the value of mix recombinant antigen in schistosomiasis diagnosis. Methods The recombinant antigens of SjC23 (HD),SjC21.7 and SjCMP10 were expressed in vitro and purified by the affinity chromatography method. The efficacies of soluble egg antigen (SEA),single recombinant antigen and mix recombinant antigen for schistosomiasis diagnosis by Enzyme-linked Immunosorbent Assay were compared. Results The diagnostic efficacy was the same when the antibody IgG of the same group sera of schistosomiasis was detected by different quantities of 2.5 ?g/ml and 7.5 ?g/ml of SEA immobilized on microplate, and their absorbency A was the same, but there was a significant difference in the diagnostic efficacies between single recombinant antigen and mix recombinant antigen when the antibody IgG of the same group sera of schistosomiasis was detected by the same quantity of single recombinant antigen or mix recombinant antigen immobilized on microplate, the absorbency A of mix antigen reacted with the sera of schistosomiasis was significant higher than that of the single recombinant. The positive rates were very similar when 39 sera of acute schistosomiasis,80 sera of chronic schistosomiasis and 27 sera of advanced schistosomiasis were detected by SEA or mix recombinant antigen by ELISA in the same time. No cross-reaction presented when 20 clonorchiasis sera were detected by the mix recombinant antigen and no false positive presented when 40 of healthy sera were detected by the mix recombinant antigen. Conclusion The schistosomiasis diagnostic method by using the mix recombinant antigen has been established, which is helpful for improving the efficacy of schistosomiasis diagnosis.
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Objective To find out the valuable early diagnostic antigen of Schistosoma japonicum. Methods The sera of rabbits were collected at different time after the rabbits were infected with cercariae of Schitosoma japonicum. The fractions of the soluble cercaria antigen (SCA), soluble adult worm antigen (AWA) and soluble egg antigen (SEA) were separated by SDS-PAGE and recognized by Western blotting with rabbits' sera of different time of post-infection. Results In Western blotting, the bands of 94, 48, 41, 40 kDa and 38 kDa of SCA appeared the earliest and were recognized by the rabbits sera of 2-week post-infection, the bands of 71 kDa and 23 kDa of SCA reacted with the rabbits sera of 3-week post-infection strongly. The bands of 71 kDa and 58 kDa of AWA appeared the earliest and were recognized by rabbits sera of 3-week post-infection. The bands of SEA reacted earliestly to the rabbits sera of 4-week post-infection were 270, 151, 73, 69, 50 kDa and 24 kDa. Conclusion The fraction antigens of 94, 71, 48, 41, 40, 38 kDa and 23 kDa of SCA, the fraction antigens of 71 kDa and 58 kDa of AWA and the fraction antigens of 270, 151, 73, 69, 50 kDa and 24 kDa of SEA could be recognized by sera of acute infected rabbits and might have potential early immuno-diagnosis value for schistosomiasis.
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Objective To develop a fast, simple immunodiagnosis assay for early diagnosis of schistosomiasis. Methods The soluble cercariae antigen(SCA) labeled colloidal dye was used as the detecting antigen for schistosomiasis. A dipstick dye immunoassay(SCA-DDIA) for early diagnosis of schistosomiasis was established. The antibodies in sera of infected rabbits in early stage of infection by SCA-DDIA were detected and compared with SEA-DDIA. The sera from people with acute and chronic schistosomiasis and other parasitic diseases and from healthy people were tested by SCA-DDIA and SEA-DDIA. Results In infected rabbits during early stage of infection, the average time of antibody detected by SCA-DDIA was 22 d , at day 30 post-infection all experimental rabbits were positive with SCA-DDIA, the detected time was earlier than that with SEA-DDIA. The sensitivity of SCA-DDIA for acute, chronic schistosomiasis japonica were 100.0% and 93.3% respectively. The specificity for healthy persons was 99.0%. The cross reaction rates with paragonimiasis westermani, clonorchiasis sinensis and fasciolopsiasis buski were 26.3%, 0 and 0 respectively. The results were similar to that by SEA-DDIA. Conclusion The SCA-DDIA is more useful for early diagnosis of schistosomiasis.
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Objective To explore the protective effect of recombinant superoxide dismutase(SOD)fusion protein against the infection of Schistosoma japonicum Chinese strain.Methods The recombinant SOD fusion protein was expressed and purified with Glutathione sepharose 4B.C57BL/6J mice were immunized with the recombinant SOD fusion protein mixed with Freund adjuvant.Four weeks after the final immunization,the mice of the experiment and control groups were challenged with(45?2)S.japonicum cercariae.All the mice were sacrificed on the forty-fifth day after the challenge to calculate the worm reduction rate and egg reduction rate,and to observe the pathologic changes of liver tissue of the mice.Results The worm reduction rate was 35.63% and the egg reduction rate was 31.17% in the experiment group.The number of granuloma in the live tissue of the experiment group was less than that of the control group,and the mean diameter of single granuloma in the experiment group reduced by 22.32% compared with that of the control group.The IgG subclass levels of IgG1,IgG2a,IgG2b were higher than those of the control group.Conclusion The recombinant SOD fusion protein has a protective effect against Schistosoma japonicum infection.
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Objective To synthesize and fusion express the predicted T-cell epitopes of Schistosoma japonicum, and analyze their immunogenicities. Methods The plus and minus oligo-nucleic acid strands of epitopes P7, P17, P18 were synthesized following their DNA sequence, respectively. The Nco I restriction enzyme sites were added to the 5′ end of epitope gene and the Xho I restriction enzyme sites were added to the 3′ end of epitope gene. The plus and minus strand of each epitope gene was annealed to form double strand DNA fragments. Then the double strand DNA fragments encoding epitope peptide were cloned into the site between Nco I and Xho I of plasmid pET32c(+) to construct recombinant plasmid which was transformed into E.coli DH5?. The recombinant plasmid containing P7, P17, P18 genes respectively was identified by PCR, restriction digestion and DNA sequencing, and then transformed into E.coli BL21 (DE3) for expressing the fusion protein. The fusion protein of peptide-thioredoxin(Trx) was expressed by inducing with IPTG and analyzed with SDS-PAGE. The fusion proteins were purified with Ni2+ column affinity chromatography. Meanwhile, the peptides P7, P17, P18 were synthesized artificially following their amino acid se-quence. By using the purified epitope peptide fusion proteins and synthesized epitope peptides, the splenic cells of C57BL/6J mice immunized with ultraviolet-attenuated cercaria of Schistosoma japonicum were stimulated respectively. The stimulation activity of fusion proteins and synthesized peptides were assayed by detecting the incorporation rate of 3 H-thymidine. Results The double strand DNA fragments of epitopes P7, P17, P18 were successfully cloned to form recombinant plasmids, all of which could express a 20 kDa fusion protein. Both the fusion protein and synthesized epitope peptides of P7 and P17 were able to stimulate the lymphocyte cells to proliferation effectively. Conclusion The peptide P7 and peptide P17 are testified as T-cell epitopes of Schistosoma japonicum.
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Objective To predict B cell epitopes in Sj22, Sj23, Sj14-3-3, Sj26 of Schistosoma japonicum with bioinformatics, and evaluate the antigenicity of these epitope proteins. Methods The complete DNA sequences of S.japonicum were predicted by BioSun system, the target B cell epitope genes were selected, cloned and expressed. The expressed fusion proteins were detected with the sera of schistosomiasis patients and health people for evaluation of their antigenicity. Results Eight B cell epitopes from four molecules of S.japonicum were predicted. The B cell epitopes of Sj22 probably located in 56-62 and 127-133 amino acids. The B cell epitopes of Sj23 probably located in 149-156 and 160-167 amino acids. The B cell epitopes of S14-3-3 probably located in 118-125 and 130-137 amino acids. The B cell epitopes of Sj26 probably located in 143-149 and 191-197 amino acids. The predicted epitope genes were cloned into pET-32c plasmid and expressed. Three of eight expressed fusion proteins of epitopes were reacted with the sera of schistosomiasis patients but not with health people. Conclusion Three epitope antigens with potential diagnosis value are determined.
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Objective To observe the molluscicidal effect of 25% suspension concentrate of niclosamide (SCN) against snail eggs and young snails. Methods The experiments with SCN and 50% wettable powder of niclosamide ethanolamide salt (WPN) against the snail eggs and young snails were carried out by the immersion method in laboratory. Results The death rates of snail eggs were both 100% in 0.25 mg/L active content of SCN and in 0.50 mg/L of WPN for 24 hours. The LC_~50(s) of SCN against Oncomelania snail eggs were 0.0506, 0.0496 mg/L and 0.0473 mg/L by immersion for 24, 48 hours and 72 hours respectively, while the LC_~50(s) of WPN were 0.1030, ~0.0962 mg/L and 0.0869 mg/L. The death rates of young snails were both 100% in 0.25 mg/L active content of SCN and WPN for 24 hours. The LC_~50(s) of SCN were 0.0625, 0.0474 mg/L and ~0.0442 mg/L for 24, 48 hours and 72 hours respectively, while the LC_~50(s) of WPN were 0.1088, 0.0825 ~mg/L and 0.0825 mg/L. Conclusion The SCN has high molluscicidal effect against Oncomelania snail in its different developmental stages: egg and young snail.[