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Objective To construct prokaryotic expression vectors for glutamate dehydrogenase(GDH)of Clostridium difficile(CD),and express recombinant GDH in Escherichia coli,and identify its antigenicityed.Methods The entire gene of GDH was cloned from ATCC43255 genome DNA.The recombinant antigens were expressed in E.coli with IPTG induction and purified by Ni-NATBeads.The antigenicity was detected using CD Qick Chek Complete dual-antigen EIA. Results Prokaryotic expression vectors of CD GDH were constructed successfully.The antigen could be identified by specific anti-GDH antibodies.Conclusion The GDH antigen can be used to prepare corresponding antibodies,which facilitate the development of immunoassay for CD GDH.
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Objective To obtain different fragments of human carboxypeptidase H,and evaluate the diagnostic application of the recombination carboxypeptidase H in detecting autoantibody.Methods The coding gene of carboxypeptidase H was ob-tained by RT-PCR.The corresponding prokaryotic expression vectors were constructed and transformed into E.coli to in-duce the expression of the recombination different fragments of carboxypeptidase H.Using these antigen fragments as the coating antigens,the enzyme-linked immunosorbent assay (ELISA)was established for the detection of carboxypeptidase H autoantibody in 95 newly diagnosed type 2 diabetes patients.Results Three fragments of human carboxypeptidase H were obtained,in which the 42~476aa fragment antigen was ideal one.Using the full-length carboxypeptidase H as coating anti-gen,the positive rate of carboxypeptidase H autoantibody was 8.42%.Conclusion Because of the favorable antigenicity,the 42~476aa fragment antigen of carboxypeptidase H could be the candidate antigen for discrimination and diagnosis of latent autoimmune diabetes in adults.
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Objective To research CpG and Al(OH)3 adjuvants enhancing immunogenicity of hepatitis C virus(HCV) recombinant ptotein combined vaccine(TIE).Methods BALB/c mice were immunized with candidate vaccine TFE using CpG,Al(OH)3,Al(OH) 3 + CpG,or freund's adjuvant(FA) as the adjuvant.Five mice were sacrificed after 10 d of the last immunization.Specific antibodies in sera were tested by enzyme-linked immunosorbent assay(ELISA).Splenic cells were isolated and levels of IFN-γ,IL-4 and cytotoxic T lymphocyte(CTL) cytotoxicity assay were messuredin vitro.The remaining mice were subcutaneouly injected with 1 × 106 SP2/0-NS3 cells on the back to investigate the protective effects.The differences of means between groups were compared by LSD-t test.Results The specific CTL activity of TFE + A1(OH) 3 + CpG group was higher than TFE + FA group and TFE + CpG group(P ＜ 0.05).The level of IFN-γsecreting cells in TFE + Al(OH)3 + CpG group was higher than that in TFE + M(OH)3 group or TFE + CpG group(P ＜ 0.05).Conclusion Combining Al(OH) 3 and CpG could enhance specific cellular immunogenicity of candidate HCV vaccine TFE.TFE + M(OH) 3 + CpG could effetively prevent the attack of tumor cell SP2/0-NS3 expressing nonstructural protein NS3 of HCV.
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Objective To design a complex hepatitis C vires(HCV)=E1 antigen,and to search its application in HCV vaccine and diagnosis test.Methods Through consulting the database and widely comparison of sequences from HCV E1 of different genotype,the representative immunodominant epitope sequences were selected from all the six genotypes.Their genes were deduced according to the preference codon in E.coli and three fragments were designed to contain the six epitopes.They were chemically synthesized and cloned into pBVIL1 vector separately.The cloned fragments were conjugated together profiting from pBVIL1's property,and then a doubled pan-DR helper T cell epitopes(PADRE)gene was inserted to form a complex expression plasmid.The transformed E.coli cells with this plasmid were cultured and induced to express recombinant protein and the antigenic activity of the product was tested.Results An expressing plasmid containing 8 epitopes from HCV genotype 1a,1b,2a,3a,4a,6a and a doubled PADRE sequences was constructed successfully and the engineering E.coli transformed with this plasmid highly expressed after inducing culture at 42℃ in an inclusion manner.The immunological activity of the purified recombinsnt multi-epitope antigen shows that:(1)It can react with a great part of sera from HCV positive patients by indirect ELISA.(2)It can induce notable specific humoral immunity in injected mice.Conclusion The novel constructed expressing plasmid and its product may be useful in study of a newly HCV vaccine as well as an antigen for HCV immunoassays.
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Objective To establish a sandwich enzyme-linked immunosorbent (ELISA) method for early diagnosis of hepatitis C. Methods Immunization of Balb/c mice with hepatitis C core antigen were prepared by genetic engineering. Mouse monoclonal antibodies (McAb) to anti-HCV-core Ag were obtained. A sandwich ELISA kit for detecting HCV-core Ag was developed by using four strains of anti-HCV-core Ag McAb. One hundred and twenty five serum specimens with increased ALT but negative for anti-HCV, anti-HIV, and anti-Tp tests were tested. Results Nine of the 125 specimens were positive for HCV-core Ag. Conclusion The double sandwich ELISA kits for detecting HCV-core Ag may be useful for the early diagnosis of hepatitis C .