梅毒螺旋体Tp92蛋白的抗原表位筛选与鉴定
发布时间：2018-08-31 12:46
【摘要】：目的：筛选和鉴定梅毒螺旋体(Treponema pallidum, Tp)Tp92蛋白的B、Th和联合B-Th细胞抗原表位，为深入探讨这些表位在梅毒表位疫苗中的作用奠定基础。 方法： (1)从GenBank获取Tp92蛋白氨基酸序列，预测其信号肽序列；采用亲水性、柔韧性、抗原指数及表面可能性方案，以IEDB和Bcepred软件分析工具综合预测Tp92蛋白的B细胞表位；以RANKPEP、SYFPEITHI软件预测综合预测Tp92蛋白的HLA-DRBl限制性Th细胞表位；根据上述结果综合预测Tp92蛋白的联合B-Th细胞表位。 (2) RP—HPLC人工合成和纯化预测的表位多肽，质谱分析和鉴定合成多肽。 (3)以梅毒患者血清(同时设健康人血清对照)，用间接ELISA鉴定预测Tp92蛋白的B细胞表位或联合B-Th细胞抗原表位。 (4)分离培养梅毒患者和正常人外周血单个核细胞(PBMC)，以预测合成的Th或联合Th-B细胞抗原表位刺激培养的PBMC，同时设ConA (阳性对照)和RPMI-1640(阴性对照)刺激，用CCK-8淋巴细胞增殖试剂盒检测培养PBMC中淋巴细胞增殖水平，鉴定Tp92中预测的T细胞表位；同时以ELISA试剂盒检测培养的淋巴细胞分泌INF-γ和IL-4的水平，，以鉴定Th细胞表位的类型。 结果： (1)综合分析计算机预测方案，筛选出P1(Tp9224-39AA)、P2(Tp92332-347AA)、P3(Tp92520-536AA)、P4(Tp92575-588AA)、P5(Tp92103-118AA)、P6(Tp92694-712AA)为Tp92候选B细胞表位；P5(Tp92103-118AA)、P6(Tp92694-712AA)、P7(Tp92668-680AA)、P8(Tp92300-313AA)、P9(Tp92396-410AA)为Tp92候选Th细胞表位；P5、P6为候选联合Th-B表位。 (2)经RP-HPLC纯化及纯度分析，合成多肽纯度均大于90%；质谱分析合成多肽分子量测定值与理论值一致。 (3) ELISA分析表明，预测的P1、P2、P3、P4、P5、P6均与梅毒患者血清反应呈阳性，而与健康人血清不反应。 (4)淋巴细胞增殖试验表明，P6、P7、P9能诱导梅毒患者淋巴细胞明显增殖，而不能诱导正常人淋巴细胞增殖；P6、P7、P9诱导梅毒患者淋巴细胞产生较高水平IFN-γ；P5、P6、P7、P8、P9均未能诱导梅毒患者淋巴细胞明显产生IL-4。 结论： 本研究初步得出以下结论： (1) P1、P2、P3、P4、P5、P6为Tp92蛋白潜在的特异性B细胞表位，尤其是P3和P6； (2) P6、P7、P9为Tp92蛋白潜在的HLA-DRBl限制性特异性Th1型细胞表位； (3) P6为Tp92蛋白潜在的特异性联合B-Th1细胞表位。
[Abstract]:Objective: to screen and identify the epitopes of (Treponema pallidum, Tp) Tp92 protein of Treponema pallidum and B-Th cell epitopes, and to lay a foundation for further study of the role of these epitopes in the vaccine of syphilis epitopes. Methods: (1) the amino acid sequence of Tp92 protein was obtained from GenBank and its signal peptide sequence was predicted. B cell epitopes of Tp92 protein were comprehensively predicted by IEDB and Bcepred software, HLA-DRBl restricted Th epitopes of Tp92 proteins were predicted by RANKPEP,SYFPEITHI software. According to the above results, the combined B-Th cell epitopes of Tp92 protein were comprehensively predicted. (2) the predicted epitope peptides were synthesized and purified by RP-HPLC. The synthetic polypeptides were analyzed and identified by mass spectrometry. (3) the B cell epitopes of Tp92 protein or the antigen epitopes of B-Th cells were predicted by indirect ELISA using syphilis patients' serum. (4) In vitro, (PBMC), of peripheral blood mononuclear cells (PBMC) from cultured syphilis patients and normal subjects were used to predict the synthesis of Th or PBMC, stimulated by Th-B cell antigen epitope. Both ConA (positive control) and RPMI-1640 (negative control) stimuli were used. CCK-8 lymphocyte proliferation kit was used to detect lymphocyte proliferation in cultured PBMC to identify T cell epitopes predicted in Tp92, and ELISA kit was used to detect the levels of INF- 纬 and IL-4 secreted by cultured lymphocytes to identify the type of Th cell epitopes. 缁撴灉锛
本文编号：2215026
[Abstract]:Objective: to screen and identify the epitopes of (Treponema pallidum, Tp) Tp92 protein of Treponema pallidum and B-Th cell epitopes, and to lay a foundation for further study of the role of these epitopes in the vaccine of syphilis epitopes. Methods: (1) the amino acid sequence of Tp92 protein was obtained from GenBank and its signal peptide sequence was predicted. B cell epitopes of Tp92 protein were comprehensively predicted by IEDB and Bcepred software, HLA-DRBl restricted Th epitopes of Tp92 proteins were predicted by RANKPEP,SYFPEITHI software. According to the above results, the combined B-Th cell epitopes of Tp92 protein were comprehensively predicted. (2) the predicted epitope peptides were synthesized and purified by RP-HPLC. The synthetic polypeptides were analyzed and identified by mass spectrometry. (3) the B cell epitopes of Tp92 protein or the antigen epitopes of B-Th cells were predicted by indirect ELISA using syphilis patients' serum. (4) In vitro, (PBMC), of peripheral blood mononuclear cells (PBMC) from cultured syphilis patients and normal subjects were used to predict the synthesis of Th or PBMC, stimulated by Th-B cell antigen epitope. Both ConA (positive control) and RPMI-1640 (negative control) stimuli were used. CCK-8 lymphocyte proliferation kit was used to detect lymphocyte proliferation in cultured PBMC to identify T cell epitopes predicted in Tp92, and ELISA kit was used to detect the levels of INF- 纬 and IL-4 secreted by cultured lymphocytes to identify the type of Th cell epitopes. 缁撴灉锛
本文编号：2215026
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