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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。
Objective To determine the epitopes of Streptococcus pneumoniae autolysin (LytA) protein CD4 T cells and CD8 T cells. Methods The sequences of MHC-Ⅰ binding peptide (CD8 T-cell epitope) and MHC-Ⅱ binding peptide (CD4 T cell epitope) of LytA protein mouse were predicted by bioinformatics methods and the corresponding peptides were synthesized. The purified rLytA protein was used to immunize mice induced by expression, purification, dialysis, endotoxin removal and quantification. The spleen and lymph node cells of each immunized group and control group were separated and compared with each artificial Candidate epitope peptides were co-cultured in vitro, and the supernatant was collected for double-sandwich ELISA to detect the production of IFN-γ in each group of cells. Splenocytes and lymph node cells of mice were stimulated with the epitope peptides screened by the primary screening and Cell flow cytometry was performed to screen out the T cell epitopes of LytA protein. Results MHC-Ⅰ binding peptides and MHC-Ⅱ binding peptides of LytA protein molecules were analyzed by a variety of methods. Totally 26 candidate MHC-Ⅰ binding peptides and 7 candidate MHC-Ⅱ binding peptides were obtained and synthesized. The rLytA protein was obtained by molecular biology technique and successfully immunized mice. The result of double sandwich ELISA showed that the peptides LⅠ4, LⅠ5, LⅠ6, LⅠ7, LⅠ11, LⅠ12, LⅠ13, LⅠ14, LⅠ25 and LⅡ3, LⅡ4, LⅡ5 The amount of IFN-γ produced by stimulation of immunized mice cells was significantly higher than that of the corresponding control mice. The cell flow cytometry was further confirmed by immunization with peptides LⅠ4, LⅠ6, LⅠ7, LⅠ11, LⅠ12, LⅠ13, LⅠ14 and LⅠ25 The percentages of double positive cells of IFN-γ and CD8 in mouse cells were significantly higher than that of control cells. The percentages of double positive cells of IFN-γ and CD4 in immunized mice after stimulated by LⅡ4 and LⅡ5 were The percentage of control mouse cells was significantly increased. Conclusion The epitopes of two CD4 T cells and eight CD8 T cells of Streptococcus pneumoniae LytA protein were identified.