Objective To obtain and characterize a novel gene of Schistosoma japonicum. Methods Looking for a novel gene through expressed sequence tags(EST),then predicting its function with the help of software on line. Finally the novel gene was cloned into an eukaryotic plasmid pEGFP N3. Results A novel gene with complete ORF was obtained. It has homogeneity with Defender Against Apoptotic Death 1 of human and pig. Conclusions EST is a good method to find new genes and analyse them with the help of software on line and characterize their function.

Objective To establish a rapid, specific and sensitive diagnostic technique for the human T. gondii infection with hematopoietic stem cell transplantation and discuss its clinical significance. Me- thods Fifty-six patients subject to hematopoietic stem cell transplantation were detected with ELISA and PCR. Results Among 56 recipients of hematopoietic stem cell transplantation, 7 were positive for T. gondii antigen and 10 were positive for SAG3 gene fragment respectively with the positive rate being 14.3 % and 17.8 % in the ELISA and PCR screening respectively. Twenty healthy people were negative for anti-Toxo antibody.Conclusion PCR is an accurate, relatively rapid, sensitive and specific method for detecting SAG3 gene of T. gondii, and can be considered a valuable additional tool for identification of T. gondii infections.

Objective To Construct the prokaryotic expression vector of the fusion gene IFN-?1b/CSPⅡ. MethodsIFN-?1b was amplified from the human genomic DNA by PCR and cloned into prokaryotic expression vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1/IFN-?1b was constructed. Circumsporozoite proteinⅡ(CSPⅡ) was amplified from the Plasmodium falciparum genomic DNA by PCR and was cloned into the prokaryotic expression vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1/CSPⅡ was constructed. IFN-?1b was cut from the recombinant plasmid pGEX-4T-1/IFN-?1b digested with BamHⅠ and EcoRⅠ and ligated with the recombinant plasmid pGEX-4T-1/CSPⅡ also digested with BamHⅠ and EcoRⅠ . The recombinant prokaryotic plasmid pGEX-4T-1/IFN-?1b/CSPⅡ was constructed. The fusion gene IFN-?1b/CSPⅡ was expressed in E.coli by IPTG. Results The prokaryotic expression vector pGEX-4T-1/IFN-?1b, pGEX-4T-1/CSPⅡ and pGEX-4T-1/IFN-?1b/CSPⅡ were identified by PCR, enzyme digestion and gene sequencing. The expressed fusion protein/IFN-?1b/CSPⅡ in E.coli was identified by SDS-PAGE and Western blot. Conclusion The prokaryotic expression vector of the fusion gene IFN-?1b/CSPⅡ was successfully constructed, which was then expressed in E.coli .

Objective To produce prokaryotic recombinant protein glyceraldehyde-3-phosphate dehydrogenase of Clonorchis sinensis (CsGAPDH), analyze its enzyme activity and immunological function. Methods The recombinant CsGAPDH was purified according to the protocol of GST?Bind~TM kit and was digested with thrombin proteinase and eluted with wash buffer. The BALB/c mice were inoculated with the purified protein. The antisera collected from the mice were used to detect the titres of IgG antibodies by ELISA, and Western blotting was used to identify the specificity of the antisera with the purified CsGAPDH. S-P immunohistochemistry method was used to confirm the expression and distribution of CsGAPDH in adult Clonorchis sinensis with the polyclonal antibodies from immunized BALB/c mice. The CsGAPDH catalytic activity was evaluated employing the conventional substrate glyceraldehydes-3-phosphate (3-GAP). Results SDS-PAGE showed a single purified protein band. Gel scanning analysis revealed that the protein purity of CsGAPDH was 90%. ELISA analysis showed an increased IgG value. S-P immunohistochemistry analysis demonstrated that the recombinant plasmid pGEX-4T-1-GAPDH expressed and distributed in muscle cell membrane of immune mice. Western blotting result suggested that CsGAPDH protein contained essential epitopes with high antigenic activities. This protein CsGAPDH could catalyzed 3-GAP with enzymatic active unit of 2 872 U min~-1ml~-1. Conclusion The recombinant protein CsGAPDH shows a proper enzymatic activity and immunogenicity.

Objective To clone the gene of autolysin(LytA) which are from different clinical strains of Streptococcus pneumoniae and express them in Escherichia coli. Methods The LytA gene was amplified by PCR from the total DNA of S.pneumoniae. Primers were designed according to the LytA gene sequence of R6. Recombinant plasmids were constructed and the sequences of different clinical strains were analyzed through method of bioinformatics. The cloned genes were expressed in E.coli and detected by SDS-PAGE. Results Complete LytA gene were amplified from all of the different clinical strains of S.pneumoniae and recombinant plasmids pGEX-4T-1-LytA were constructed successfully. After comparing the sequence of DNA and supposed protein, we find some differences. Induced by IPTG, LytA gene was expressed effectively in E.coli Jm109. Result of SDS-PAGE showed that the molecular weight of expressed protein was 62 kD, the same as calculated. Conclusions The sequences encoding LytA from different clinical strains of S.pneumoniae were cloned, the recombinant plasmids pGEX-4T-1-LytA was constructed successfully. Sequence analysis showed that there have difference among the gene and amino acid sequences of LytA from different clinical strains. Further studies should be focused on whether the difference contributes to activity of autolysin and the drug-resistance of S.pneumoniae.

Aim To search the effective malaria mixed gene vaccine and to explore the immune response of vaccinated host. The recombinant pcDNA3-EBA175/HRP Ⅱ was injected alone or mixed with pcDNA3-Pfs25 of Plasmodium falciparum into mice by intramuscular route. The muscle of injected parts were treated previously, e.g. injecting 50μl of 0. 5％bupivacaine into the muscle of left latter leg, with 2mm depth. To observed the changes of IgG antibody value, the splenic lymphocyte proliferation, the ratio of CD4+/CD8+ subgroups and NK cell killing activity. After injecting recombinant pcDNA3-EBA175/HRP Ⅱ alone or mixed with pcDNA3-Pfs25 into mice by intramuscular route, it showed that sera IgG value increased, the splenic T lymphocyte proliferation stimulated by specific antigen of Plasmodium falciparum increased, the ratio of CD4+/CD8+ decreased and NK cell activity increased. Enhanced immune injection could improve host's immune reaction.It is suggested intramuscular injection is an effective immune route, and mice inoculated with coding two gene recombinant alone or that mixed with sexual stage gene recombinant could all induce increased humoral and cellular immune response, and NK cell activity.

Objectve To detect whether the CTP(phosphocholine cytidylyltransferase) gene was expressed in the asexual erythrocytic stages of Plasmodium falciparum (FCC 1/HN )by using the RT - PCR and to construct eukaryotic expression vector of CTP. Method The erythrocytic stage parasites of Plasmodium falciparum were cultured as described by Trager and Jensen. RNA from erythrocytic stage parasite was extracted by using Trizol reagent. The complete genes coding for CTP gene isolates FCCI/HN were amplified by reverse transcriptase -polymerase chain reaction(RT- PCR). CTP gene was cloned into eukaryotic expression vector pcDNA3. Results CTP encoding gene was amplified from the erythrocytic stages of Plasmodiumfalciparum (FCC 1/HN) and eukaryotic expression vector of CTP was constructed. Conclusion CTP gene was expressed in the erythrocytic stages of Plasmodium falciparum (FCC 1/HN) and eukaryotic expression vector of CTP was successfully constructed.

Objective To obtain the recombinant IgE-dependent histamine-releasing factors of Schistosoma japonicum and Clonorchis sinensis (rSjHRF and rCsHRF) and to study the effect of recombinant HRFs to induce histamine release from sensitized rat mast cells. Methods The complete coding regions of SjHRF and CsHRF were cloned separately, and the recombinant plasmids were respectively transformed and expressed in BL21 cells. The soluble recom-binant rSjHRF and rCsHRF were purified. Aliquots of the mast cells obtained from the lungs of OVA-immunized rats were separately incubated with rSjHRF and rCsHRF and the released histamine was measured by the OPT spectrofluorometric procedure. The dose-dependent curves and the kinetics of histamine release induced by rSjHRF and rCsHRF were prepared. Results The recombinant plasmids pET-30-rSjHRF and pET-30-rCsHRF were constructed successfully and the purified soluble recombinant proteins rSjHRF and rCsHRF were obtained by affinity chromatography. rSjHRF and rCsHRF induced histamine release from sensitized mast cells in a dose-dependent manner. At the concentration of 150 mg/L, the average rate of histamine release from sensitized mast cells induced by rSjHRF and rCsHRF were 49.78% and 32.63%, respectively. Histamine release increased with prolonged reaction time and the maximal release occurred at 35min. Conclusion The recombinant parasite-originated IgE-dependent HRFs show an effect of inducing histamine release from sensitized mast cells, suggesting that this protein would play a role in type I hypersensitivity in hosts with parasitic infections.

Objective To construct prokaryotic recombinant plasmids of transcriptional co-activator (TC) gene of Clonorchis sinensis, express and purify the recombinant protein and analyze its biological function. Methods A pair of primers was designed according to the known sequence of TC gene. The TC gene fragment was amplified by PCR. After purification and digestion with BamHⅠ and SalⅠ , the TC gene was connected to the prokaryotic expression vectors, pGEX-4T-1 and pET30a(+). By cloning target gene into these vectors, pGEX-4T-1 and pET30a(+), prokaryotic recombinant plasmids of TC gene were constructed and transferred into E.coli BL21. The positive expressed recombinants were detected by SDS-PAGE and Western blotting. Immobilized metal (Ni 2+ ) chelation affinity chromatography was used to purify His-TC produced by the expression of the recombinant protein pET30a(+)-TC. Results The recombinant plasmids, pGEX-4T-1-TC and pET30a(+)-TC, were constructed successfully. SDS-PAGE testified that the molecular weight of the recombinant protein was correct. Western blot analysis of GST-TC recombinant protein testified that the recombinant protein could be recognized by immunized rabbit serum, which means the protein is GST-immune active and the clone can express recombinant Clonorchis sinensis antigen. After affinity chromatography of the pET-TC protein, there was only one protein band with expected size on the SDS-PAGE gel. Conclusion The TC gene was screened from cDNA library of adult Clonorchis sinensis, cloned, expressed and purified. The purified protein of TC gene will be of importance for further research on the biological function of the gene.

Objective To recognize and identify the arginase(ARG)gene of Schistosoma japonicum(Sj),and to study its protection potential as a vaccine.Methods The 5'-end of the ARG gene from the Sj cercariae cDNA library was amplified by nested-PCR and the sequence was identified by bioinformatics.The complete coding sequence(CDS)was cloned into pET30a(+)vector,and a recombinant SjARG protein(rSjARG)was expressed,purified and used to raise antibodies.ARG's activity as an enzyme was tested by ornithine-ninhydrin reaction.Western blotting was used to compare the immunologic characteristics of rSjARG with that of the native one in Sj adult worm.Indirect immunofluorescence assay was used to immunolocalize it.For evaluating the protection potential of rSjARG,mice were immunized by the recombinant protein and challenged by cercariae of S.japonicum.Results The CDS length of the SjARG novel gene was identified as 1095bp.rSjARG showed enzyme activity and the same immunologic characteristics with the native arginase in adult worm.SjARG located in the genital organ and gut of both sexes.The worm reduction rate and egg reduction rate in rSjARG group were 55.8% and 48.8% respectively,higher than that of the rSj26GST group(28.6% and 6.89% respectively).Conclusion SjARG gene was identified,which shows a higher protection than the Sj26GST.