梅毒螺旋体TprF蛋白B细胞表位的预测与鉴定

目的 预测和鉴定梅毒螺旋体(Tp) 膜蛋白TprF氨基端保守区(TprF^N)的优势B细胞表位,为深入研究梅毒多价表位疫苗提供依据。方法 从GenBank获取TprF^N的氨基酸序列,采用Mobyle、ABCpred和IEDB在线软件综合分析预测TprF^N的B细胞表位并人工合成多肽;表达重组蛋白TprF^N并经Western blot鉴定后免疫兔,获取血清并测定抗体效价;以TprF^N免疫兔血清、梅毒患者血清(设正常人血清和正常兔血清为阴性对照),间接ELISA测定预测的7条人工合成的B细胞表位多肽的免疫反应性和特异性。结果 软件综合预测TprF^N的P1 (43-62AA)、P2(57-71AA)、P3(81-88AA)、P4(89-103AA)、P5(125-138AA)、P6(231-251AA)、P7(268-279AA)可能为B细胞表位;表达一可溶性蛋白,WB鉴定为目的 蛋白,其免疫抗体效价为1∶12 800以上;ELISA结果显示,预测表位P1、P3与TprF^N免疫兔血清及梅毒患者血清均呈阳性反应,而与对照血清均不反应。结论 P1、P3为TprF潜在的优势B细胞表位。

Prediction and identification of B-cell epitopes of Treponema pallidum repeat protein F

To predict and identify the dominant B-cell epitopes of conserved region of Treponema pallidum repeat protein F (TprF^N) and provide the basis for development of polyvalent epitope-based syphilis vaccine, the amino acid sequence of TprF^N was obtained from GenBank and analyzed with comprehensive meta-analysis Mobyle, ABCpred and IEDB online software. The peptides containing predicted epitopes were artificially synthesized. To obtain and measure the titers of antibodies against TprF^N, New Zealand rabbits were immunized with recombinant protein TprF^N expressed in E. coli and identified by Western blot (WB) . Sera from TprF^N-immunized rabbits, syphilis patients, and normal human and normal rabbits were used to determine the immunoreactivity and specificity of 7 predicted peptides of TpF^N by indirect ELISA. Comprehensive meta-analysis of online software showed that P1 (43-62AA), P2(57-71AA), P3(81-88AA), P4(89-103AA), P5(125-138AA), P6(231-251AA) and P7(268-279AA) might be the B-cell epitopes. A protein was expressed in a soluble form and identified as TpF^N by WB. The ELISA indicated that P1 and P3 were active with TprF^N-immunized rabbit sera and syphilis patient sera but not with negative control sera. These results indicate that P1 and P3 are the potential dominant B-cell epitopes.