ABSTRACT
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.
ABSTRACT
Objective To provide the candidate antigens for immunological diagnosis by analyzing the expression of nu -cleoprotein ( NP) of Ebola virus. Methods BioSun software was used to predict the NP epitopes. The bridging-PCR was used to synthesize the NP gene. The pBVIL1 vector was used to clone and express the NP gene. Results The 360-739 aa of NP was confirmed to be the dominant antigen by BioSun software. The recombinant NP dominant antigen was expressed in E.coli with molecular weight of 58 ×103.The specificity of ELISA based on recombinant NP was 99.24% (130/131) in negative samples. Conclusions The dominant NP antigen can be potentially used for developing Ebola virus diagnostic reagent.
ABSTRACT
Bcel-2 family proteins (Bcl-x(L), Bcl-2, Mel-1 etc.) are key regulators of some life processes, including apoptosis and autophagy. They are currently considered as promising targets for developing new anti-tumor therapies. In our study, the human Bcl-2/Bcl-x(L) chimeric gene and the human/mouse Mel-1 chimeric gene were designed and cloned, and the prokaryotic expression vectors for expressing glutathione S-transferase (GST) fusion proteins and histidine tag fusion proteins were constructed respectively. These two proteins as well as the GST-Bcl-x(L) fusion protein were all successfully expressed in E. coli and subsequently purified. In addition, we measured the binding of these Bcl-2 family proteins to the Bid BH3 peptide by fluorescence polarization-based assay. The dissociation constants (Kd) obtained by us were in general agreement with the data reported in literature. The Kd values of all three proteins with or without the GST tag were almost identical. All these results validate the biological functions of these Bcl-2 family proteins obtained by us. These proteins can be used in the experimental screening of small-molecule regulators of Bcl-2 family proteins in vitro.
Subject(s)
Humans , Escherichia coli , Genetics , Metabolism , Fluorescence Polarization , Methods , Glutathione Transferase , Genetics , Myeloid Cell Leukemia Sequence 1 Protein , Proto-Oncogene Proteins c-bcl-2 , Genetics , Recombinant Fusion Proteins , Genetics , bcl-X Protein , GeneticsABSTRACT
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.