肺炎链球菌自溶酶(LytA)蛋白T细胞表位的预测、筛选及确定

Predication and identification of T cell epitopes of Streptococcus Pneumoniae LytA protein

目的确定肺炎链球菌自溶酶(LytA)蛋白CD4 T细胞及CD8 T细胞的表位。方法通过生物信息学方法预测肺炎链球菌LytA蛋白分子小鼠的MHC-Ⅰ结合肽(CD8 T细胞表位)和MHC-Ⅱ结合肽(CD4 T细胞表位)序列并人工合成相应肽段;经诱导表达、纯化、透析、去内毒素、定量等步骤得到纯化的rLytA蛋白用以免疫小鼠;分离每只免疫组小鼠和对照组小鼠的脾及淋巴结细胞并分别与每条人工合成的候选表位肽段体外共培养,取培养上清进行双夹心ELISA检测每组细胞IFN-γ的产生情况;用初步筛选到的表位多肽刺激小鼠的脾及淋巴结细胞并进行细胞内染色及细胞流式技术检测,筛选出LytA蛋白分子T细胞表位。结果利用多种方法分析了LytA蛋白分子的MHC-Ⅰ结合肽及MHC-Ⅱ结合肽,经整理得到候选MHC-Ⅰ结合肽26条,候选MHC-Ⅱ结合肽7条,并进行了人工合成。利用分子生物学技术获得了rLytA蛋白并成功免疫了小鼠;双夹心ELISA结果初步提示:肽段LⅠ4、LⅠ5、LⅠ6、LⅠ7、LⅠ11、LⅠ12、LⅠ13、LⅠ14、LⅠ25以及肽段LⅡ3、LⅡ4、LⅡ5刺激免疫小鼠细胞产生的IFN-γ量比相应的对照小鼠显著升高;细胞流式技术进一步确定:经肽段LⅠ4、LⅠ6、LⅠ7、LⅠ11、LⅠ12、LⅠ13、LⅠ14及LⅠ25刺激后,免疫小鼠细胞中IFN-γ和CD8双阳性的细胞百分比较对照小鼠细胞的百分比显著升高;经肽段LⅡ4和LⅡ5刺激后,免疫小鼠细胞中IFN-γ和CD4双阳性的细胞百分比较对照小鼠细胞的百分比显著升高。结论确定了肺炎链球菌LytA蛋白分子2个CD4 T细胞及8个CD8 T细胞的表位。

Autolysin （LytA） is a major candidate antigen for vaccine against Streptococcus pneumoniae infection. To identify CD4 T cell and CD8 T cell epitopes of LytA, we predicted and synthesized both MHC- I binding peptides （CD8 T cell epitopes） and MHC- Ⅱ binding peptides （CD4 T cell epitopes） by bioinformatics methods. Purified rLytA was obtained through molecular biology methods and used to immunize mice. Then cells of spleen and lymph node were separated from immunized mice and negative control mice and cultured with synthesized peptides respectively, and then the cell supernate was collected to detect IFN-γ secretion by double sandwich ELISA. Every primly selected epitope were used to stimulate cells, and then intracellular cytokine staining and flow cytometric analysis were performed to identify T cell epitopes. Double sandwich ELISA showed 9 of MHC- I peptides and 3 of MHC- 11 peptides could stimulated significant higher IFN-γ secretion, as compared with control group; Flow cytometric analysis suggested that after stimulation by 8 of MHC- I peptides, the percentage of CD8 and IFN-γ double positive cells were significant higher in immunized mice than that in negative control group; while the percentage of CD4 and IFN-γ double positive cells were significant higher in immunized mice than that in negative control group after been stimulated by 2 of MHC-Ⅱ peptides. In conclusion, we identified amino acid seauence and location of two CD4 T cell epitopes and eight CD8 T cell epitopes of Streptococcus pneumoniae LytA protein which will help to develop epitope-based vaccine.