【摘要】：目的：篩選和鑒定梅毒螺旋體(Treponema pallidum, Tp)Tp92蛋白的B、Th和聯(lián)合B-Th細(xì)胞抗原表位，為深入探討這些表位在梅毒表位疫苗中的作用奠定基礎(chǔ)。 方法： (1)從GenBank獲取Tp92蛋白氨基酸序列，預(yù)測(cè)其信號(hào)肽序列；采用親水性、柔韌性、抗原指數(shù)及表面可能性方案，以IEDB和Bcepred軟件分析工具綜合預(yù)測(cè)Tp92蛋白的B細(xì)胞表位；以RANKPEP、SYFPEITHI軟件預(yù)測(cè)綜合預(yù)測(cè)Tp92蛋白的HLA-DRBl限制性Th細(xì)胞表位；根據(jù)上述結(jié)果綜合預(yù)測(cè)Tp92蛋白的聯(lián)合B-Th細(xì)胞表位。 (2) RP—HPLC人工合成和純化預(yù)測(cè)的表位多肽，質(zhì)譜分析和鑒定合成多肽。 (3)以梅毒患者血清(同時(shí)設(shè)健康人血清對(duì)照)，用間接ELISA鑒定預(yù)測(cè)Tp92蛋白的B細(xì)胞表位或聯(lián)合B-Th細(xì)胞抗原表位。 (4)分離培養(yǎng)梅毒患者和正常人外周血單個(gè)核細(xì)胞(PBMC)，以預(yù)測(cè)合成的Th或聯(lián)合Th-B細(xì)胞抗原表位刺激培養(yǎng)的PBMC，同時(shí)設(shè)ConA (陽(yáng)性對(duì)照)和RPMI-1640(陰性對(duì)照)刺激，用CCK-8淋巴細(xì)胞增殖試劑盒檢測(cè)培養(yǎng)PBMC中淋巴細(xì)胞增殖水平，鑒定Tp92中預(yù)測(cè)的T細(xì)胞表位；同時(shí)以ELISA試劑盒檢測(cè)培養(yǎng)的淋巴細(xì)胞分泌INF-γ和IL-4的水平，，以鑒定Th細(xì)胞表位的類型。 結(jié)果： (1)綜合分析計(jì)算機(jī)預(yù)測(cè)方案，篩選出P1(Tp9224-39AA)、P2(Tp92332-347AA)、P3(Tp92520-536AA)、P4(Tp92575-588AA)、P5(Tp92103-118AA)、P6(Tp92694-712AA)為Tp92候選B細(xì)胞表位；P5(Tp92103-118AA)、P6(Tp92694-712AA)、P7(Tp92668-680AA)、P8(Tp92300-313AA)、P9(Tp92396-410AA)為Tp92候選Th細(xì)胞表位；P5、P6為候選聯(lián)合Th-B表位。 (2)經(jīng)RP-HPLC純化及純度分析，合成多肽純度均大于90%；質(zhì)譜分析合成多肽分子量測(cè)定值與理論值一致。 (3) ELISA分析表明，預(yù)測(cè)的P1、P2、P3、P4、P5、P6均與梅毒患者血清反應(yīng)呈陽(yáng)性，而與健康人血清不反應(yīng)。 (4)淋巴細(xì)胞增殖試驗(yàn)表明，P6、P7、P9能誘導(dǎo)梅毒患者淋巴細(xì)胞明顯增殖，而不能誘導(dǎo)正常人淋巴細(xì)胞增殖；P6、P7、P9誘導(dǎo)梅毒患者淋巴細(xì)胞產(chǎn)生較高水平IFN-γ；P5、P6、P7、P8、P9均未能誘導(dǎo)梅毒患者淋巴細(xì)胞明顯產(chǎn)生IL-4。 結(jié)論： 本研究初步得出以下結(jié)論： (1) P1、P2、P3、P4、P5、P6為Tp92蛋白潛在的特異性B細(xì)胞表位，尤其是P3和P6； (2) P6、P7、P9為Tp92蛋白潛在的HLA-DRBl限制性特異性Th1型細(xì)胞表位； (3) P6為Tp92蛋白潛在的特異性聯(lián)合B-Th1細(xì)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- 緯 and IL-4 secreted by cultured lymphocytes to identify the type of Th cell epitopes. 緇撴灉錛

本文編號(hào)：2215026