结核分枝杆菌PPE家族蛋白CD4^+ T细胞泛宿主表位的计算机预测及其γ干扰素释放实验研究

Computational Prediction of Promiscuous CD4^+ T-cell Epitopes from PPE Protein Family of Mycobacterium tuberculosis and IFN-γ Release Assays

目的利用计算机预测结核分枝杆菌PPE家族蛋白MHC-II类分子限制性CD4+T细胞泛宿主表位,并利用γ干扰素释放实验进行体外验证。方法利用网上数据库提取PPE家族全部蛋白序列。使用生物信息学软件包括Signal P4.1,Secretome P 2.0,DAS和Net MHCII2.2等分析工具预测、筛选出MHC-II类分子限制性CD4+T细胞泛宿主表位。最后利用γ干扰素释放实验进行合成肽段的体外实验验证其刺激激活CD4+T细胞的能力。结果发现了34个泛宿主表位多肽,其中21个来自于PPE8蛋白,其余来自于PPE12、PPE21和PPE62蛋白。γ干扰素释放实验并未发现预测出的泛宿主表位多肽可刺激大量的CD4+T淋巴细胞产生IFN-γ的分泌,但来自于PPE8蛋白的两个泛宿主表位多肽可刺激少量的CD4+T淋巴细胞产生反应。结论这些计算机预测出来的泛宿主表位多肽,尤其是来自PPE8蛋白的泛宿主表位多肽,可能是下一步亚单位疫苗或诊断性标志物的重要候选抗原。但仍需进一步扩大样本量进行验证。

Objective To present a computational approach to screen and select promiscuous MHC class II restricted CD4＋T cell epitopes in the PPE protein family of M. tuberculosis and evaluate the level of protection in IFN-γ release assays. Methods Through database access to protein sequences of the PPE protein family,and some bioinformatics programs including Signal P4. 1 server, Secretome P 2. 0 server, DAS server, and Net MHCII2. 2 server were used to predict human promiscuous MHC class II restricted CD4＋T cell epitopes.Then IFN-γ release assay was used to confirm the ability of certain epitopes to stimulate and activate CD4＋T cell. Results Thirty-four promiscuous epitope peptides were identified; 21 from PPE8 and others from PPE12,PPE21 and PPE62. IFN-γ release assay did not provide evidence of a significant IFN-γ＋CD4＋T cell response to these promiscuous epitope peptides. Two epitope peptides from PPE8 protein could activate weak immune responses. Conclusion Prediction analysis showed that these promiscuous epitope peptides,especially from PPE8 protein,may be important targets in subunit vaccines or diagnostic antigens against MTB. Future research need to base upon larger sample.