钩端螺旋体抗原表位预测、重组、表达及免疫原性分析

Prediction, recombination and expression of Leptospiral epitopes and analysis of its immunogenicity

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 epitopes from two of outer membrane proteins in Leptopspira by bioinformation, and to recombinant, express and analyze recombinant epitopes＇ immunogenicity. Methods The prediction programs are ProPred and ANTIGENIC. Recombinant epitope gene was constructed by PCR. The PCR product was then cloned to construct recombinant vector. The gene was induced to express fusion protein in E. coli host strain BL21（DE3） and the fusion protein was purified. Anti-serum of BALB/c mice immunized with fusion protein was tested by MAT to evaluate the fusion protein＇s immunogenicity. Results Two peptides functioning both as MFIC binding peptides and B cell epitopes were predicted from each of the two outer mambrane proteins. There was no frame-shift mutation nor base substitution in the sequence. The fusion protein was obtained with the purity over 90%. Results of MAT showed that the anti-serum titer of fusion protein was 75.79, while the anti-senun titer of GST was 10.62. Conclusion The results confirmes that the recombinant epitope has certain immunogenicity, and it＇s potential for study on recombining epitopes and subunit vaccine in related protein.