金黄色葡萄球菌IsdA蛋白B细胞线性抗原表位的预测与鉴定

Prediction and evaluation of B cell epitopes of the IsdA protein of Staphylococcus aureus

目的预测金黄色葡萄球球菌Newman株IsdA蛋白B细胞线性抗原表位,并对所预测B细胞表位的免疫反应性进行初步鉴定。方法以金黄色葡萄球球菌Newman株IsdA蛋白为对象,采用NCBI提供的蛋白质查询程序确定IsdA蛋白的氨基酸序列,然后使用网络在线预测软件SOPMA分析IsdA蛋白质二级结构;使用IEDB网络服务器预测IsdA蛋白B细胞线性表位和Th细胞表位;使用VectorNTI10.0软件分析IsdA蛋白序列中易形成B细胞表位的氨基酸-缬氨酸、亮氨酸及半胱氨酸所在的位置。综合以上分析结果获得所预测B细胞线性表位。以预测表位序列为参考,人工合成表位多肽,通过肽间接ELISA法验证所预测表位与IsdA多克隆抗体血清的免疫反应性。结果 IsdA存在潜在的B细胞线性抗原表位,所预测表位区分别位于N末端P_(120-140)、P_(181-200)、P_(210-230)和P_(300-320),间接ELISA检测rIsdA多克隆抗体血清中存在着可与P_(210-230)、P_(300-320)结合的组分。结论 IsdA蛋白潜在的B细胞线性表位位于N末端P_(210-230)和P_(300-320)区域,具有与特异性血清结合的能力,为金黄色葡萄球菌的表位疫苗研发奠定了基础。

Objectives To predict B cell epitopes of the IsdA protein of the Newman strain of Staphylococcus aureus and verify the immunoreactivity of the predicted epitopes. Methods Programs from the NCBI were searched for the sequence of the IsdA protein of the Newman strain of S.aureus.The secondary structure of the IsdA protein was determined with the software SOPMA.B cell epitopes and Th cell epitopes were predicted using online tools of the IEDB.The location of 3amino acids（valine,leucine,and cysteine）was analyzed and these sites were identified in the epitopes.The sites where valine,leucine,and cysteine readily constituted an epitope were analyzed with the software VectorNTI10.0.All of the above results were comprehensively analyzed and the B cell epitopes of IsdA were verified.Epitope peptides were synthetized based on predicted B cell epitopes and the immunoreactivity of epitope peptides was verified using indirect ELISA.Plates were coated with peptide antigens so that they would bind to polyclonal antibodies against rIsdA. Results Based on the predicted secondary structure of IsdA,IEDB scores,and the location of valine,leucine,and cysteine,the B cell linear epitope of IsdA was potentially located in N-terminal regions of P120-140,P181-200,P210-230,and P300-320.An indirect ELISA assay indicated that epitope peptides P210-230 and P300-320 bind to polyclonal antibodies against rIsdA（t=0.66±0.03,0.72±0.05,P〈0.05）.Conclusion B cell epitopes of the IsdA protein were located at P210-230 and P300-320,and these epitopes bind to polyclonal antibodies against rIsdA.These findings have laid the foundation for the study of epitope vaccines against S.aureus.