Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A region of feline immunodeficiency virus (FIV)/Glasgow-8 external envelope glycoprotein (env) incorporating the third and fourth variable regions (V3/V4) was cloned, inserted into the pGEX vector and expressed in Escherichia coli to yield milligram quantities of the recombinant polypeptide as a fusion protein with glutathione S-transferase. The fusion protein V3/V4GST was used in lymphocyte proliferation assays, where it consistently caused peripheral blood lymphocytes from naive cats to proliferate in a dose-dependent manner. Other FIV fusion proteins produced under identical conditions (V5GST and p24GST) and glutathione S-transferase alone did not cause proliferation in this system. The monoclonal antibody vpg15, which has been shown to block infection of susceptible cells in vitro, did not decrease the response to V3/V4GST. Human peripheral blood lymphocytes did not proliferate in response to V3/V4GST.
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PMID:A recombinant feline immunodeficiency virus envelope fusion protein stimulates peripheral blood lymphocytes from naive cats to proliferate in vitro. 133 93

The gp85 envelope glycoprotein of Epstein-Barr virus (EBV) has a role in the molecular mechanism of infection, enabling fusion between the viral and host cell envelopes, a role in common with the homologous gH glycoproteins in other herpesviruses. A glutathione S-transferase bacterial fusion protein (GST85N-S) was generated, containing 178 amino acids from the C terminus of gp85 and including a known gp85 linear epitope. A panel of EBV-positive human antisera contained no antibodies to linear epitopes presented on the purified GST85N-S protein, indicating that primary protein structure in this region of gp85 is not a B cell target. This bacterial fusion protein was used to raise a rabbit monospecific polyclonal antiserum capable of detecting gp85 in a Western blot. The majority of recombinant baculovirus-expressed gp85 obtained from cell extracts prepared with SDS appeared on Western blots as heterogeneous high M(r) protein aggregates and consistently included 84K, 81K and 70K bands. Recombinant gp85 aggregation was increased by boiling the sample prior to gel electrophoresis. The 84K and 81K proteins were completely sensitive to endoglycosidase H treatment, indicating that these glycosylated species did not undergo further post-translational processing. Immunofluorescence studies revealed that recombinant gp85 was not transported to the insect cell surface. It reacted only with antibodies recognizing denatured gp85 and not with antibody to native gp85. Therefore expression of the gene encoding gp85, BXLF2, alone in the baculovirus expression system is insufficient for the synthesis of a correctly transported, processed, folded and antigenically native form of recombinant gp85.
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PMID:Expression of the Epstein-Barr virus envelope fusion glycoprotein gp85 gene by a recombinant baculovirus. 752 63

Defined segments of the gag polyprotein and transmembrane envelope glycoprotein from Maedi-visna virus were expressed as glutathione S-transferase fusion proteins in Escherichia coli and evaluated singly and in combination for use in an enzyme-linked immunosorbent assay (ELISA). Two hundred sixty field serum specimens from 15 sheep flocks were tested in parallel with recombinant and whole-virus antigens, and the relative sensitivities and specificities of the recombinant antigens were calculated. When the recombinant gag and transmembrane proteins were used in combination, a sensitivity of 97.4% and a specificity of 99.4% relative to whole-virus antigen were observed, indicating the utility of these proteins in diagnostic testing.
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PMID:Evaluation of two recombinant Maedi-visna virus proteins for use in an enzyme-linked immunosorbent assay for the detection of serum antibodies to ovine lentiviruses. 854 46

Complementary DNA encoding the ORF5 gene of a Quebec reference isolate (IAF-Klop) of porcine reproductive and respiratory syndrome virus (PRRSV) was cloned into the prokaryotic expression vectors pGEX-4T and pET21a to produce ORF5-glutathione S-transferase and ORF5-polyhistidine fusion proteins. Five hybridoma cell lines producing monoclonal antibodies (MAbs) to the 25 kDa viral envelope glycoprotein (GP5) were obtained from BALB/c mice immunized with the affinity chromatography-purified GST-ORF5 fusion protein. The polypeptide specificity of these anti-PRRSV MAbs, belonging to the IgG1 isotype, was confirmed by Western immunoblotting assays with recombinant and native viral proteins, and by radioimmunoprecipitation using [35S]methionine-labelled concentrated extracellular virus. All these MAbs showed virus-neutralizing (VN) activity, with VN titres ranging from 1:32 to 1:128. Two MAbs (IAF-1B8 and IAF-8A8) reacted with similar titres with the modified live attenuated vaccine strain ATCC VR-2332, but all five failed to react to the prototype European strain, the Lelystad virus, in VN and indirect immunofluorescence tests. The results obtained suggest that these five anti-PRRSV MAbs are directed to serotype-specific linear neutralizing epitopes which are not affected by the absence of carbohydrate residues.
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PMID:Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants. 926 81

The ORF5-encoded major envelope glycoprotein (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) is one of the three major structural proteins of this virus. While some porcine convalescent sera and monoclonal antibodies directed against GP4 and GP5 have the capacity to neutralize the virus in vitro, the protein specificity of porcine neutralizing sera has not yet been established. DNA immunization with a plasmid encoding GP5 of PRRSV, under the control of a human cytomegalovirus promoter, induced anti-GP5-specific neutralizing antibodies in pigs and BALB/c mice. The GP5 protein specificity of neutralizing sera was confirmed by immunoblotting and ELISA. Peripheral blood mononuclear cells obtained from DNA-vaccinated pigs underwent blastogenic transformation in the presence of E. coli-expressed recombinant ORF5-encoded protein, indicating the specificity of the cellular immune response to GP5. Following a massive intratracheal challenge with the virulent IAF-Klop strain of PRRSV, DNA-vaccinated pigs were protected from generalized viraemia and the development of typical macroscopic lung lesions that were observed in unvaccinated, virus-challenged controls, as well as in pigs that were immunized with E. coli-expressed GST-ORF5 recombinant fusion protein. Interstitial pneumonitis and broncho-alveolitis were remarkably milder in DNA-vaccinated animals. These results suggest that the GP5 of PRRSV is a good candidate for a subunit recombinant-type vaccine.
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PMID:Immune response in pigs vaccinated with plasmid DNA encoding ORF5 of porcine reproductive and respiratory syndrome virus. 960 13

The objective of the present study was to evaluate the importance of genomic and antigenic variations which may have affected the major envelope glycoprotein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) isolates responsible for outbreaks in Quebec and Ontario, in comparison with the modified-live U.S. vaccine strain (MLV) and the European prototype strain from Lelystad (LV). Nucleotide sequence analyses of the open reading frame (ORF)5 genes showed that all of the isolates studied were heterogenous, amino acid (aa) identities varied from 88 to 99% with the MLV strain, and between 51 and 54% with the LV strain. The aa substitutions were randomly scattered across the protein, although one region between residues 26 and 39 was found to correspond to a hypervariable region which involved 0 to 3 potential N-glycosylation sites. The ORF5 encoded products of 5 of these isolates, including the MLV and LV strains, were expressed in E. coli as recombinant proteins fused to the glutathione S-transferase (GST) protein and used to raise hyperimmune anti-ORF5 sera in rabbits. The reactivity patterns of strain-specific hyperimmune anti-ORF5 sera and a panel of 4 monoclonal antibodies directed against the ORF5 gene product of the Quebec IAF-Klop strain of PRRSV, indicated that GP5 of field isolates also underwent antigenic variations. The data suggest that neutralizing epitopes, independent of conformation and glycosylation, are also associated with antigenic variability of the GP5 of PRRSV.
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PMID:Genomic and antigenic variations of porcine reproductive and respiratory syndrome virus major envelope GP5 glycoprotein. 968 45

Antagonism is the ability of a modified antigenic peptide (altered peptide ligand, APL) to prevent CD4 T cell activation by the original peptide. Here we show that antagonistic activity can be conferred to peptides of HIV envelope glycoprotein gp120 and reverse transcriptase p66 by adding flanking polypeptide sequences at the C or at the N terminus by genetic engineering, rather than by introducing substitutions by synthesis. The glutathione S-transferase (GST)-peptide system has been used to produce molecules that display the peptide at the appropriate end of the GST carrier. When the gp120 peptide 191-205 (pep24) was expressed at the C terminus of GST (GST-24), antigenicity of specific human CD4 T cells was maintained. In contrast, when the peptide was expressed at the N terminus of GST (24-GST), antigenicity was abolished and antagonistic activity was introduced. Similar results were obtained with a p66-derived peptide at the C terminus of the GST carrier. Antagonism was (1) specific; proliferation of a CD4 T cell line from the same donor responding to the envelope glycoprotein of another retrovirus, HTLV-1, was not affected; (2) reversible; proliferative response was rescued in T cells exposed to antigen-presenting cells (APC) pulsed with the antagonist; (3) dominant; T cells cultured with APC pulsed with the agonist and with APC pulsed with the antagonist did not proliferate. The carrier could be cleaved by proteolysis while the antagonistic activity was preserved. Thus a minimal sequence that confers antagonistic activity can be engineered or synthesized with peptides to antagonize undesired CD4 responses as an alternative to the use of APL.
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PMID:Antagonistic activity of HIV-1 T helper peptides flanked by an unrelated carrier protein. 1035 98

Enzyme-linked immunosorbant assays (ELISAs) were developed for the detection of antibodies against the major envelope glycoprotein (G(L)) of equine arteritis virus (EAV). A 6-Histidine tagged recombinant protein expressing the complete G(L) ectodomain (G(L)-6His), a glutathione-S-transferase recombinant protein expressing amino acids 55-98 of G(L) (G(L)-GST) and an ovalbumin-conjugated synthetic peptide representing amino acids 81-106 of G(L) (G(L)-OVA) were used as diagnostic antigens. An ELISA procedure was developed and optimised for each antigen. The G(L)-OVA and G(L)-6His assays showed the greatest specificity while the G(L)-GST assay was slightly more sensitive that the G(L)-OVA and G(L)-6His assays; results based on the analysis of 50 virus neutralisation positive and 50 virus neutralisation negative sera. The G(L)-OVA ELISA was selected for further evaluation since it was simpler to use than ELISAs based on recombinant antigens and did not suffer from background reactivity. The final sensitivity and specificity of the G(L)-OVA ELISA were 96.75 and 95.6%, respectively, results based on the analysis of 400 virus neutralisation positive and 400 virus neutralisation negative sera. It also detected EAV antibody (100% efficiency) in seropositive shedding stallions and, in ponies infected experimentally with the UK93 isolate of EAV, the appearance of virus neutralising antibodies and G(L)-OVA ELISA-specific immunoglobulins coincided.
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PMID:Development and evaluation of ELISA procedures to detect antibodies against the major envelope protein (G(L)) of equine arteritis virus. 1106 17

Monoclonal antibody 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asia, American and European strains, but antibodies responses to ELDKWA-epitope in HIV-1 infected individuals were very low. Based on the epitope-vaccine strategy suggested by us, a recombinant glutathione S-transferase (GST) fusion protein (GST-MELDKWAGELDKWAGELDKWAVDIGPGRAFYGPGRAFYGPGRAFY) as vaccine antigen containing three repeats of neutralizing epitope ELDKWA on gp41 and GPGRAFY on gp120 was designed and expressed in Escherichia coli. After vaccination course, the recombinant multi-epitope vaccine could induce high levels of predefined multi-epitope-specific antibodies in mice. These antibodies in sera could bind to both neutralizing epitopes on gp41 peptide, V3 loop peptide and recombinant soluble gp41 (aa539-684) in ELISA assay (antisera dilution: 1:1,600-25,600), while normal sera did not. Moreover, these antibodies in sera could recognize the CHO-WT cells which expressed HIV-1 envelope glycoprotein on the cell surfaces, indicating that the predefined epitope-specific antibodies could recognize natural envelope protein of HIV-1 though these antibodies were induced by recombinant multi-epitope-vaccine. These experimental results suggested a possible way to develop recombinant multi-epitope vaccine inducing multi-antiviral activities against HIV-1.
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PMID:Recombinant multi-epitope vaccine induce predefined epitope-specific antibodies against HIV-1. 1227 May 53

We have developed a widely applicable method to construct epitope-peptide gene for epitope-vaccine strategy recently. In this study, we wanted to know whether the predefined spacers between epitopes on a recombinant epitope-peptide impacted the production of epitope-specific antibodies. The neutralizing epitope ELDKWA on the C-domain of HIV-1 gp41 was defined by the monoclonal antibody (mAb) 2F5 with broad neutralizing activity. We constructed three recombinant ELDKWA-epitope-peptides with different spacers between epitopes. The recombinant epitope-peptide GST-K8, GST-S8 and GST-R8 were bearing eight copies of ELDKWA-epitope with amino acid spacer GS, GSGGGGS and RS, respectively. GST-K8 and GST-S8 could induce high titer of ELDKWA-epitope-specific antibodies, much better than GST-R8. Besides, both antibodies could recognize the recombinant soluble gp41 and the transfected CHO-WT cells that stably express HIV-1 envelope glycoprotein on the cell surfaces. These experimental results indicated that the spacer GSGGGGS and GS were feasible in constructing a recombinant epitope-vaccine.
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PMID:Predefined spacers between epitopes on a recombinant epitope-peptide impacted epitope-specific antibody response. 1562 74


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