目的：揭示突变蛋白SEC2（T20L／G22E）超抗原活性较野生型SEC2显著增强的潜在机制。方法：WST-1法检测突变蛋白SEC2（T20L／G22E）在体外刺激小鼠脾淋巴细胞增殖活性，进一步应用流式细胞术和荧光定量PCR技术分别检测CD4＋和CD8＋T淋巴细胞增殖、特异性VβT淋巴细胞增殖以及相关细胞因子mRNA丰度变化。结果：与野生型SEC2相比，突变蛋白SEC2（T20L／G22E）能够在体外显著刺激小鼠脾淋巴细胞增殖，但二者具有相似的特异性VβT淋巴细胞增殖谱，并且SEC2（ T20L／G22E）能够较rSEC2激活更多携带有Vβ5.3、Vβ8.1及Vβ8.3的T 淋巴细胞；进一步体内实验发现 SEC2（T20L／G22E）能够较rSEC2激活更多外周血和脾脏中CD4＋和CD8＋T淋巴细胞增殖及相关细胞因子的表达。结论：突变蛋白SEC2（T20L／G22E）超抗原活性增强的机制可能是用亮氨酸和谷氨酸替代原来20和22位上的氨基酸后提高了SEC2（T20L／G22 E）与特异性TCR-Vβ的亲和力，进一步激活更多的SEC2特异性T淋巴细胞，最终引起T淋巴细胞亚型的显著性增殖，同时诱导更多与抗肿瘤、增殖相关的细胞因子的表达。%Objective:To investigate the improved superantigen activity of SEC2(T20L/G22E) compared with recombinant staphylococcal enterotoxins C 2 ( rSEC2 ).Methods: The proliferation of spleen lymphocytes and T-cell subpopulations induced by rSEC2 and SEC2(T20L/G22E) were examined by WST-1 and flow cytometry separately,and the gene expression of cytokines and Vβspecificities were quantified by real-time PCR.Results: WST-1 and Flow cytometry assays showed that the superantigen activity of SEC2(T20L/G22E) was improved due to enhanced T-cell stimulating potency,resulting in massive activation of T-cells,particularly CD4+and CD8+T-cells.Quantitative real-time PCR assay showed that despite similar Vβspecificities induced by rSEC 2 and SEC2 (T20L/G22E),the quantities of activated T-cells bearing specific Vβwere different,and SEC2(T20L/G22E) could stimulate more gene expression of associated cytokines simultaneously.Conclusion: The results strongly suggested that the increased SEC 2 ( T20L/G22 E)-TCR-binding affinity contributed to more T-cells activation and cytokine release ,which elicit powerful immune activition.
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