B族链球菌C5a肽酶蛋白表位的克隆、表达和免疫学分析

目的应用生物信息学方法预测B族链球菌C5a肽酶蛋白表位，结合基因工程手段进行表位重组、表达和免疫原性分析。方法用预测程序ProPred和ANTIGENIC预测B族链球菌C5a肽酶蛋白的表位，应用PCR技术扩增出编码该表位基因片段，克隆PCR产物构建重组质粒，测序验证。在大肠杆菌BL21（DE3）中诱导表达融合蛋白。表达的蛋白经质谱分析和Western blot鉴定。纯化该融合蛋白并免疫C57／BL小鼠，萃取GBS表面蛋白，双向琼脂扩散试验检测抗体水平。结果在SCPB中预测到1个既具有MHC结合肽特性又具有B细胞表位特征的肽段。重组和表达了这一肽段，质谱得出与SCPB蛋白的相似性分数为79，Western-blot证实能与抗SCPB的抗体反应，纯化后融合蛋白纯度〉90％。动物实验证实融合蛋白能产生特异性的抗GBS抗体。结论重组表位具有一定免疫原性。为相关蛋白的毒力机制研究和亚单位疫苗等方面的研究打下了良好的基础。

Prediction, recombination and expression of streptococcal C5a peptidase epitope and its immunogenicity

Objective To predict epitopes of streptococcal C5a peptidase by bioinformatics, and to recombinant, express and analyze recombinant epitope and its immunogenicity. Methods The prediction programs are ProPred and ANTIGENIC. Recombinant epitope gene was constructed by PCR. The PCR product was then cloned into a constructed recombinant vector. The gene was induced to express fusion protein in E. coli host strain BL21 (DE3) and the fusion protein was identified by pepude mass fingerprinting (PMF) analysis and Western blot. Anti serum of C57/BL mice immunized with purified fusion protein was tested by double immunodiffusion to evaluate the fusion protein's immunogenicity. Results The peptide functioning both as MHC binding peptides and B cell epitopes was predicted from SCPB. The probability based mouse score of C5a peptidase was 79. The recombinant protein could specially react with antibody against SCPB. The fusion protein was obtained with the purity over 90% . Results of double immunodiffusion showed that the antiserum of fusion protein could specially react with surface proteins isolated from GBS strain. Conclusion The results confirm that the recombinant epitope has certain immunogenicity, and its potential for the study on recombining epitopes and subunit vaccine in related protein.