title: Development of a procedure for the direct cloning of T-cell epitopes using bacterial expression systems.
abstract: Although single bacterial recombinant antigens have been used successfully to stimulate individual T-cell clones and elicit recall responses in peripheral lymphocytes, the broader use of molecular cloning systems for the identification of autoantigens recognised by the cellular arm of the immune system has met with only limited success. In a systematic approach to address this problem, a series of bacterial expression vectors were examined for their potential use as cloning vectors to elicit a proliferative response in vitro from a non-obese diabetic (NOD) mouse T-cell clone which recognises the immunodominant ovalbumin epitope (aa 323-339). The use of the vector pRSET, which produces a hexa-histidine tagged fusion protein, was confounded by non-specific responses to bacterial protein contaminants. pGEX, which generates a glutathione-S-transferase hybrid, avoided this problem but suffered from the disadvantage that a universally applicable purification procedure for the hybrid antigen could not be easily developed. A practical screening protocol was developed using the pUEX expression system (beta-galactosidase hybrid) and purification based upon electroelution of the hybrid protein from purified inclusion bodies subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This system can be used to screen expression libraries for the detection of T-cell epitopes provided that the T-cell clones give low background responses to irrelevant pUEX recombinant proteins. Low abundance antigens may be obtained using this system in combination with subtractive hybridisation to construct cDNA libraries enriched in the target antigen.
authors.affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, UK.
authors.forename: P I
authors.initials: PI
authors.lastname: Neophytou
authors.forename: P
authors.initials: P
authors.lastname: Ozegbe
authors.forename: D
authors.initials: D
authors.lastname: Healey
authors.forename: R
authors.initials: R
authors.lastname: Quartey-Papafio
authors.forename: A
authors.initials: A
authors.lastname: Cooke
authors.forename: J C
authors.initials: JC
authors.lastname: Hutton
mesh_terms: D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics* / Q000302:isolation & purification; D002999:Clone Cells / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods*; D000939:Epitopes / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology*; D005822:Genetic Vectors / Q000737:chemistry* / Q000276:immunology*; D005982:Glutathione Transferase / Q000235:genetics; D006639:Histidine / Q000235:genetics; D008213:Lymphocyte Activation; D051379:Mice; D016688:Mice, Inbred NOD; D010047:Ovalbumin / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013601:T-Lymphocytes / Q000737:chemistry* / Q000276:immunology; D014373:Tuberculin / Q000276:immunology; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D000945:Antigens, Differentiation, T-Lymphocyte; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D014373:Tuberculin; D006639:Histidine; D010047:Ovalbumin; D005982:Glutathione Transferase; D001616:beta-Galactosidase