DEVELOPMENT OF A PROCEDURE FOR THE DIRECT CLONING OF T-CELL EPITOPES USING BACTERIAL EXPRESSION SYSTEMS

Although single bacterial recombinant antigens have been used successf ully to stimulate individual T-cell clones and elicit recall responses in peripheral lymphocytes, the broader use of molecular cloning syste ms for the identification of autoantigens recognised by the cellular a rm of the immune system has met with only limited success, In a system atic approach to address this problem, a series of bacterial expressio n vectors were examined for their potential use as cloning vectors to elicit a proliferative response in vitro from a non-obese diabetic (NO D) mouse T-cell clone which recognises the immunodominant ovalbumin ep itope (aa 323-339). The use of the vector pRSET, which produces a hexa -histidine tagged fusion protein, was confounded by non-specific respo nses to bacterial protein contaminants, pGEX, which generates a glutat hione-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 screeni ng protocol was developed using the pUEX expression system (beta-galac tosidase hybrid) and purification based upon electroelution of the hyb rid protein from purified inclusion bodies subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This system c an 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 b e obtained using this system in combination with subtractive hybridisa tion to construct cDNA libraries enriched in the target antigen.