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目的应用生物信息学方法预测两种钩端螺旋体外膜蛋白的表位,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LipL32和Om-pL1的表位,应用PCR技术合成重组表位基因片段,克隆PCR产物构建重组质粒,测序验证。在BL21(DE3)中诱导表达融合蛋白。纯化该融合蛋白,免疫BALB/c小鼠,显微镜凝集试验(MAT法)测定抗体效价。结果在LipL32和OmpL1中各预测到2个既具有MHC结合肽特性又具有B细胞表位特征的肽段。PCR合成的重组表位基因序列中没有出现移码和碱基置换。纯化后融合蛋白纯度>90%。融合蛋白产生的抗体效价为75.79,融合头(运载蛋白)抗体效价为10.62。结论重组表位具有一定免疫原性。为相关蛋白的表位重组和亚单位疫苗等方面的研究打下了良好的基础。
OBJECTIVE: To predict the epitopes of two kinds of leptospiral outer membrane proteins by bioinformatics methods and to carry out epitope recombination, expression and immunogenicity analysis by means of genetic engineering. Methods ProPred and ANTIGENIC were used to predict the epitopes of LipL32 and Om-pL1. PCR was used to synthesize the recombinant epitope fragments. The PCR products were cloned and sequenced. The expression of the fusion protein was induced in BL21 (DE3). The fusion protein was purified, BALB / c mice were immunized, and antibody titers were determined by the microscope agglutination test (MAT method). Results Two of the predicted peptides in LipL32 and OmpL1 were both MHC-binding and B-cell epitopes. There was no frameshift and base substitution in the PCR-synthesized recombinant epitopes. Purified fusion protein purity> 90%. The fusion protein produced an antibody titer of 75.79 and the fusion head (carrier protein) antibody titer was 10.62. Conclusion The recombinant epitope has some immunogenicity. Lay a good foundation for the research on epitope reorganization and subunit vaccine of related proteins.