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Objective To clone and express a high mobility group box 1(HMGB1)protein of Schistosoma japonicum(Main-land strain)and analyze its function. Methods The DNA fragment of open reading frame encoding Sj HMGB1 protein was ampli-fied 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 recombi-nant 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 ca- pacity 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 pro-tein 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 calculat-ed respectively. Results 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 SjHMGB1protein 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 immu-nized 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 abun-dantly 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 effec-tive immune protection against S. japonicum. Conclusion The gene encoding HMGB1 from S. japonicum and the soluble recombi-nant 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.
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Objective To optimize the conditions of the expression of fusion protein GST-SjMP10 and to evaluate the value of fusion protein GST-SjMP10 for diagnosis of schistosomiasis.Methods The optimal concentration of IPTG for the expression of fusion protein GST-SjMP10 was chosen in inducing the expression of GST-SjMP10 with different concentration of IPTG,and the soluble fusion protein GST-SjMP10 was identified by SDS-PAGE.The fusion protein GST-SjMP10 was purified by chromatographic affinity with glutathione Sepharose 4B gel.The sensitivity and specificity of purified fusion protein GST-SjMP10 for diagnosis of schistosomiasis were determined by enzyme linked immunosorbent assay(ELISA)to detect the IgG antibody in sera from the patients with acute schistosomiasis,advanced schistosomiasis and clonorchiasis as well as healthy subjects.Results Most of the expressed fusion protein GST-SjMP10 was in soluble status when the concentration of IPTG was reduced to 0.1 mmol/L and the fusion protein GST-SjMP10 could be purified by chromatographic affinity.The positive rate of anti-GST-SjMP10 antibody in the sera from the patients with acute and chronic schistosomiasis japonica was 97.5% and 96.7% respectively.No cross reactivity of the fusion protein GST-SjMP10 was found in the detection for the sera from clonorchiasis patients,and no false positive was found in the detection for the sera of healthy subjects.Conclusion The fusion protein GST-SjMP10 was expressed successfully and showed high sensitivity and specificity for the diagnosis of schistosomiasis japonicum.
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<p><b>OBJECTIVE</b>To identify the egg antigens related to the formation of hepatic granulomas and fibrosis of Schistosomiasis japonica.</p><p><b>METHODS</b>The egg cDNA library of Schistosoma japonicum (S. japonicum) was constructed and screened by immunological methods with the pooled sera of advanced schistosomiasis patients. The inserted foreign DNA fragments of positive clones were sequenced. The sequence data were analyzed using Wdnasis 2.5 and compared with Genebank data using blast software.</p><p><b>RESULTS</b>Eighty-one clones containing recombinant DNA fragments were obtained from the egg cDNA library of S. japonicum by immunological screening. The DNA sequences of all clones belonged to the miracidial antigen family. The longest cDNA fragment was 1604 bp, which contained an open reading frame of 351 bp, which encoded a protein of 1 2913.35 daltons.</p><p><b>CONCLUSION</b>The cDNA sequence of the miracidial antigen of S. japonicum (Chinese strain) was obtained for the first time.</p>
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Animals , Antigens, Helminth , Genetics , Base Sequence , China , DNA, Complementary , Ovum , Schistosoma japonicum , Genetics , Allergy and ImmunologyABSTRACT
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 evaluate the molluscicidal effect of colistin E on Oncomelania hupensis. Methods The molluscicidal effect of colistin E on O. hupensis was checked by using the immersion method and spraying method. The toxicity of colistin E on fish was observed by using the toxic test. Results The snail mortality of each group was 100% when the snails were immersed in the colistin E solutions at a concentration of 5. 0, 1. 0 g/L for 24, 48 h and 72 h separately. When the snails were immersed in the colistin E solution at a concentration of 0. 5 g/L for 24, 48 h and 72 h, the death rates were 95% , 100% and 100% respectively; when the snails were immersed in the colistin E solution at a concentration of 0. 1 g/L for 24, 48 h and 72 h, the mortality was 90%, 95% and 100% respectively; when the snails were immersed in the colistin E solution at a concentration of 0. 01 g/L for 24, 48 h and 72 h, the mortality was 70%, 86% and 100% respectively. The snail mortality by the spraying method in a dose of 35, 70, 140 mg/m2 of colistin E was 60%, 100% and 100%. The result of toxic test showed that the toxicity of colistin E on fish was low. Conclusion Colistin E is an effective molluscicide, and is worthy of investigation further.
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Objective To screen the bacteria and its components which are toxic to Oncomelania hupensis. Methods The samples of Oncomelania hupensis on the point of death and the soil around the snails were collected. The bacteria existing in the snails and soil were isolated and identified by using regular methods. After being fermented, the toxicity of the bacterium components including the ferment supernatant, split products and bacteria to the snail were tested by the toxicity test. Results Totally, 104 strains of bacteria were isolated from the snails and soil samples, which included Gram positive cocci or bacilli, Gram negative cocci or bacilli. The fermenting supernatant, splitting products and 10 strains of bacteria showed different level of toxicity against the snail respectively. All the fermenting supernatant of bacterium PY1-1, PY7-2, PY3-5, PY19-3, PY2-2-2, PY8-2-2, PY16-1 could kill more than 50 percent of snails. Conclusion The bacteria which are poisonous to snails have been isolated that make a good basis for identifying single toxic component against snails.
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Objective To establish rSAG1-IgM-ELISA with purified rSAG1 fusion protein for immunodiagnosis of toxoplasmosis. Methods The rSAG1 fusion protein was purified by Ni 2+ column. The ELISA plate was coated with different concentrations of rSAG1, reacted with pooled positive and negtive human sera. Goat anti-human IgM conjugated to horseradish peroxidase was used as the second antibody. The appropriate detecting condition of the rSAG1-IgM-ELISA assay was determined by orthogonal experiment. The reproducibility, sensitivity and specificity of the assay were assessed. Thirty-five IgM-positive and 57 IgM-negative human sera detected by the imported IgM-ELISA kit were detected with the rSAG1-IgM-ELISA. Results The purity of rSAG1 was above 90%. The appropriate detecting condition was that the coated rSAG1 was 2 5 ?g/ml, the human serum was in 1∶100 dilution, and the second antibody was in 1∶4000 dilution. The coefficient of variation (CV) value of IgM-positive and IgM-negative pooled sera were 13 8% and 7 7% respectively. The inhibition rate of the assay was 62 0% The positive correspondence rate and negative correspondence rate were 82 9% (29/35) and 91 2% (52/57) respectively,the total correspondence rate was 88 0%, compared with the imported IgM-ELISA kit. Conclusions The rSAG1-IgM-ELISA has high sensitivity and specificity, and good correspondence rate with the imported IgM-ELISA kit. It indicates that rSAG1-IgM-ELISA has potential value for early diagnosis of toxoplasmosis.
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Objective To express the cytosolic superoxide dismutase(SOD)of Schistosoma japonicum and analyze its antigenicity.MethodsThe DNA sequence of Schistosoma japonicum gene of cytosolic SOD was amplified by reverse transcription polymerase chain reaction(RT-PCR)and subcloned into the expression vector pGEX-4T-3 to construct a recombinant plasmid Sj SOD-pGEX-4T-3.This recombinant plasmid was transformed into component cell of E.coli BL21(DE3).The fusion protein of GST-SOD was expressed by inducing with IPTG and purification by affinity chromatography.The specific antiserum was prepared by immunizing the BALB/c mouse with GST-SOD fusion protein,and the immnuogenicity of GST-SOD was investigated by Western blot analysis.ResultsThe gene of cytosolic SOD was amplified successfully and subcloned into expression vector pGEX-4T-3 forming the recombinant expression plasmid Sj SOD-pGEX-4T-3.The fusion protein GST-SOD was expressed after the recombinant containing Sj SOD-pGEX-4T-3 being induced by IPTG.Immunizing the BALB/c mouse with the fusion protein GST-SOD produced high titer specific antiserum of 1∶12800 and the fusion protein GST-SOD was recognized by sera of severe infection rabbits.ConclusionsThe cytosolic SOD has been expressed successfully and has a preferable immunogenicity.
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A kind of colloidal blue dye(D-l)used as a staining reagent for immunoassay was first selected from the dyes produced in China in this study. The optimun condition for labelling the dye onto sheep antihuman IgG antibody was explored. The dye-labelled antibody could react and stain with relative antigens. The minimal concentration of human IgG protein could be detected with the labelled dye by Dot Double Antibody Sandwich Assay was 20-10ng/ ml. 61 of 62 sera samples of schistosomiasis japonica were positive,the positive rate was 98.4%. Just 1 out of 30 healthy people's sera sambles was positive,the false positive rate was 3.3%. All of 40 Fasciolopsiasis sera samples were negative. The results were similar to that of Dot-ELISA. The activity of antibody labelled with colloidal dye could be maintained for 1 week in room temperature and be presered in lyophilized condition for a longer period. The assay was less expensive,simple to conduct and required no special equipment. It suggested that the Dot Colloidal Dye Immunoassay(DIA)might have potential value for use in schisto-somiasis endemic areas.
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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 analyze the full length cDNA sequence of Schistosoma japonicum egg miracidia genes harboring signal sequence.Methods The gene specific primers were designed and synthesized according to S.japonicum egg miracidia cDNA fragment containing signal sequence identified by signal sequence trapping method previously. The 5′and 3′ end cDNA fragments of each egg miracidia cDNA fragment harboring signal sequence were amplified by nest PCR using the first strand cDNA of S.japonicum as the template. The specific PCR fragments were cloned by TA clone method and sequenced. The full length cDNA sequence of each gene with signal sequence was constructed by comparing the cDNA sequence identified with signal sequence trapping method and the 5′ end sequence, the 3′ end sequence and deleting the overlapping fragments. The splicing model between mRNA of signal sequence and one of mature portions of S.japonicum egg miracidia gene was checked by analyzing the genomic DNA sequence structure of some genes with signal sequence. Results The 5′ and 3′ end cDNA fragments of sixteen among thirty cDNA fragment with signal sequence were amplified successfully, and their DNA sequences were determined. The full length cDNA sequences of sixteen egg miracidia genes were obtained by sequence matching and splicing. The results of deduced amino acid analysis found that the signal peptide of gene SjP4001 was the same to the one of SjP1531 and the signal peptide of gene SjP1183 was similar to the one of gene SjP3742. It confirmed that different genes could share the same or similar signal peptide. The data of S.japonicum genomic DNA sequence analysis showed that the S.japonicum could obtain its signal sequence by alternative splicing model or trans-splicing model. Conclusions The full length cDNA sequences of sixteen S.japonicum egg miracidia genes with signal sequence have been defined, it indicated that the S.japonicum egg miracidia genes could get its signal sequence by alternative splicing model or trans-splicing model was found in this study.
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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 investigate the value of recombinant fusion protein (GST-HD)of the large hydrophilic domain (HD) of 23 kDa membrane protein of Schistosoma japonicum with the Glu-tathione-S-transferase (GST) of S. japonicum for early diagnosis of schistosorniasis. Methods The rabbits were infected with cercariae of S. japonicum Chinese mainland strain (300 per one). The rabbits' sera before infection and after being infected at different time were collected. The antibodies IgG(s) against recombinant GST-HD and SEA were measured respectively by ELISA to observe the rabbits' immune reaction status to GST-HD and SEA at different time after being infected with the cercariae. At the same time, 90 serum samples of patients with acute schistosorniasis and 30 samples of healthy persons were checked with GST-HD to evaluate its value for early diagnosis of schistosorniasis. Results At the 17th, 21st and 24th day after infection, the positive rates of antibody IgG of rabbits sera against GST-HD were 42.85%, 92.80% and 100.00% respectively, but the positive rates of antibody IgG against SEA were 14. 28% , 50. 00% and 84.60% respectively. The sensitivity of GST-HD for detecting early schistosome infection was higher than that of SEA significantly. The predictive values of positive and negative of GST-HD for detecting acute schistosorniasis was 98. 89% and 96.67%,respectively, and the diagnostic efficacy was 98. 33%. Conclusion The recombinant GST-HD fusion protein has high early diagnostic value for schistosorniasis.
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Objective To screen the mimic antigen epitopes of the triose phosphate isomerase of Schistosoma japonicum Chinese strain (SjC TPI) and investigate their immunogenicity. Methods The random phage peptide library (PH^D^ 12) was screened with the purified antibody(IgG) against SjC TPI to get the positive phage which contained the mimic antigen epitopes of SjC TPI, and the immuno characterization of the mimic antigen epitopes were investigated. Results Two mimic antigen epitopes (M1, M2) of SjC TPI were obtained. The immuno sera of mice (Kunming strain) against the positive phages could recognize both the SjC TPI and the protein of the positive phages. The DNA sequencing data showed no homology between the sequences of the deduced amino acid of the two mimic antigen peptides and the amino acid of SjC TPI. Conclusion The two mimic antigen epitopes of SjC TPI obtained are imitative epitopes of the configuration antigen of SjC TPI.
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Objectives 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. Methods 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, SalI), 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. Results 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. Conclusion The miracidial antigen from eggs of Schistosoma japonicum was expressed successfully.
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Objective To study the expression of nitric oxide synthase (NOS)mRNA in the gonad of Oncamelania hu-pensis in different temperature. Methods The snails were cultured at temperature of 0℃ , 15 ℃and 25℃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). Results The target genes of NOS were detected in the gonad of snails. The level of expression of snail NOS mRNA in 25℃ group and 0℃group was significantly higher than that in the control group( P 0.05). Conclusion 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.
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Objective To observe the protective immunity induced by the nucleic acid vaccine of 21.