| Superantigens are extremely potent activators of T cells. In contrast toconventional antigens, superantigens needn't be processed by APCs, and canbind outside the classical antigen-binding groove of major histocompatibilitycomplex (MHC) class II and Vβ chain of TCR by intact protein form. Sominim superangitens can activate a large proportion (5%-20%) of T cells.Because the strong T cells activation potency, superantigens have beeninvestigated extensively as therapeutic agents in tumor therapy. People wantto borrow the potency of superantigens to elevate the antitumorimmunoreaction of human bodies, and the results of experiments have provedthe feasibility and achieved good effects. However, although exposure ofmature T cells to superantigen generally induces a marked proliferativeresponse in vivo, this often followed by antigen-specific tolerance. Suchtolerance reflects either anergy or clonal elimination. Such property ofsuperantigen strongly limits its antitumor therapy effect. Staphylococcal entertoxin A (SEA) which excreted by staphylococcus, is the most extensively investigated superantigen and also the only one which have been used in the clinical I trials on superantigen antitumor therapy. In the study, we constraucted the prokaryotic expression vector of SEA and gained SEA protein with biological activity. The SEA polyclonal antibody was successfully prepared, and the property of T cell anergy induced by SEA was studied.1. SEA gene was cloned from the genome of FRI 100, the standard SEA production strain cells, by PCR, and its sequence was identical to the report in GeneBank. The analysis of protein encoded by SEA gene showed that the first 24aa segment was a signal peptide. The signal peptide is a helicalmembranous region with high hydrophobicity, which influences the prokaryotic expression ability very much. So the expression plasmid of 702 bp which encode SEA mature protein were constructed and expressed in E coli. The obtained protein was isolated and purified, then we got the purified SEA protein and the animal experiment showed that it had good biological activity. The rabbit was immunized with the denaturalized SEA protein, and the antiserum was obtained and purified by salting out. ELISA showed the titer of antiserum was higher than 1:1,000,000. The results of Western blot and imrnunofluorescence experiments indicated that the antiserum could specifically bind with SEA2. The in vitro SEA stimulating mice spleen lymphocytes proliferation experiment showed that in a certain range of SEA concentration, the extent of lymphocytes proliferation was dependent on the concentration of SEA. When the concentration of lymphocytes was 1 × 107/mL, 1 μg/mL was the optimum stimulating concentration. The results of peripheral blood lymphocytesmensuration after SEA intraperitoneal injection showed that mice injected with 10 μg SEA had a marked increase of their peripheral blood lymphocytes (p<0.05). The peak was around the third day after SEA injection, then the lymphocytes reduced gradually and returned to the normal level on the fourteenth day; whereas mice injected with 1 μg or 0.1 μg SEA did not show the increase of their peripheral bood lymphocytes. In the experiment of induction of T cells anergy by SEA administered in vivo, C57BL/6 mice were injected intrperitoneally with SEA, then at different day after SEA injection the spleen lymphocytes were isolated and were resitmulated with optimum concentration of SEA (1 μg/mL) in vitro. The results showed: compared with control, on the ninth and fourteenth day after SEA injection, the proliferations of mice spleen lymphocytes restimulated with SEA reduced markedly (p<0.05), however on the third day, the proliferation did not decrease, but increased (p<0.05). This kind of change in lymphocytes reactive ability with SEA restimulation was not related with the dose of SEA intraperitoneal injection, since the lymphocytes from mice injected with 10 μg, 1 ug or 0.1 ug all have showed markedly reduced proliferative capacity. When the concentration of SEA restimulation concentration increased, the anerty T cells showed enhanced proliferation ability (p<0.05). The experiment of the influence of IL-2 in SEA-induced T-cell anergy indicated that, compared with control, anergic T cells had higher proliferation ablibility with exogenous IL-2 (p<0.05), which had no relationship with the dose of first SEA injection. Furthermore, the proliferation ability could be enhanced when cooperating SEA with exogenous IL-2 (p<0.05).In this study, the SEA gene was cloned and expressed, and the SEA protein with biological activity was obtained. The anti-SEA antiserum was...
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