肺炎链球菌表面粘附素A蛋白抗原表位的预测筛选及确定  

Predication,Selection,and Identification of Antigen Epitopes of Streptococcus Pneumoniae Protein PsaA

作　　者：古文雅 梁雪清[2] 麦璟莹[3] 马长玲[1] 袁竹青[1] 

机构地区：[1]广州医科大学病原生物学与免疫学教研室,广东广州510182 [2]广州医科大学2012级生物技术专业本科生,广东广州510182 [3]广州医科大学形态学实验中心,广东广州510182

出　　处：《中山大学学报（医学科学版）》2015年第1期55-64,共10页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(81072491);广州市高校科技基金(10A173)

摘　　要：【目的】确定肺炎链球菌表面粘附素A(Psa A)蛋白抗原的B细胞、CD4 T细胞和CD8 T细胞表位。【方法】通过生物信息学方法预测肺炎链球菌Psa A蛋白分子小鼠B细胞线性表位、MHC-Ⅰ结合肽(CD8 T细胞表位)及MHC-Ⅱ结合肽(CD4 T细胞表位)并人工合成相应肽段;克隆重组质粒BL21/p ET/Psa A并得到纯化的r Psa A蛋白用以免疫小鼠;分离每只免疫组和对照组小鼠的血清分别与人工合成的候选B细胞表位肽段进行间接ELISA检测,筛选B细胞表位;分离小鼠脾及淋巴结细胞并分别与人工合成的T细胞候选表位肽段体外共培养,取上清进行双夹心ELISA检测IFN-γ的量进行初步筛选。用初筛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检测,确定Psa A蛋白分子T细胞表位。【结果】分析Psa A蛋白分子的B细胞表位及MHC-Ⅰ结合肽、MHC-Ⅱ结合肽;得到候选B细胞表位肽10条,MHC-Ⅰ结合肽21条,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r Psa A蛋白并成功免疫了小鼠。间接ELISA结果显示:肽段PB2、PB6,PB7与r Psa A免疫小鼠的血清发生反应,其OD450nm读数比相应的阴性对照小鼠显著升高;双夹心ELISA结果初步提示:肽段PⅠ10、PⅠ14、PⅠ17、PⅠ18、PⅠ21以及肽段PⅡ2、PⅡ5、PⅡ6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。细胞流式结果进一步确定:经肽段PⅡ2、PⅡ5刺激后,免疫小鼠细胞中IFN-γ和CD4双阳性细胞百分比较对照小鼠细胞显著升高;经肽段PⅠ14、PⅠ17、PⅠ21刺激后,免疫小鼠细胞中IFN-γ和CD8双阳性细胞的百分比较对照小鼠细胞显著升高。【结论】确定了肺炎链球菌Psa A蛋白分子的3个B细胞线性表位;2个CD4 T细胞表位;3个CD8 T细胞表位。【Objective】 To identify B cell,CD4 T cell and CD8 T cell antigen epitopes of Streptococcus Pneumoniae protein Psa A.【Methods】 Liner B cell epitopes,MHC-Ⅰbinding peptides（CD8 T cell epitopes） and MHC-Ⅱbinding peptides（CD4 T cell epitopes） were predicted by bioinformatics methods and synthesized.Recombinant BL21/p ET/Psa A was cloned and r Psa A was purified to immunize mice.Serum was collected from each of immunized mice and negative control mice then used to perform indirect ELISA with synthesized peptides to identify B cell epitopes.Cells of spleen and lymph node were separated from every mouse and cultured with each of synthesized peptides respectively,then cell supernate was collected to detect IFN-γ secretion by double sandwich ELISA for T cell epitopes selection.Cells were stimulated by every primely selected epitopes then intracellular cytokine staining and flow cytometric analysis were performed to confirm T cell epitopes.【Result】 Several bioinformatics methods were used topredict epitopes of Psa A and 10 B cell epitopes,21 MHC-Ⅰbinding peptides and 7 MHC-Ⅱbinding peptides were synthesized.r Psa A was obtained and immunized mice successfully.Indirect ELISA showed that peptide PB2 、PB6 and PB7 reacted with immunized mice serum and the OD450 nm value were significant higher than that of the negative mice.Double sandwich ELISA showed that IFN-γconcentration that stimulated by peptide PⅠ10,PⅠ14,PⅠ17,PⅠ18,PⅠ21,and PⅡ2,PⅡ5,PⅡ6 was significant higher than its negative control group.Flow cytometric analysis confirmed that after been stimulated by P Ⅱ 2,PⅡ 5,the percentage of CD4 and IFN-γ double positive cells were significant higher from immunized mice than that from negative control group;the percentage of CD8 and IFN-γ double positive cells were significant higher from immunized mice than that from negative control group after been stimulated by PⅠ14,PⅠ17,and PⅠ 21.【Conclusion】 We identified amino acid sequence and location of three liner B cell ep

关 键 词：肺炎链球菌 表面粘附素A 抗原表位 

分 类 号：R392[医药卫生—免疫学]