中华皮肤科杂志 ›› 2018, Vol. 51 ›› Issue (5): 352-357.doi: 10.3760/cma.j.issn.0412-4030.2018.05.007

• 论著 • 上一篇    下一篇

梅毒螺旋体体内诱生抗原Tp0462的表达鉴定及在临床血清学诊断中的应用评价

刘文1,邓美霞1,张晓红2,赵铁2,罗茜1,赵飞骏3,尹卫国4   

  1. 1. 南华大学病原生物研究所/特殊病原体防控湖南省重点实验室
    2. 南华大学病原生物学研究所
    3. 湖南省衡阳市 南华大学医学院病原生物学研究所
    4. 汕头大学医学院附属粤北人民医院检验科
  • 收稿日期:2017-03-27 修回日期:2017-10-08 出版日期:2018-05-15 发布日期:2018-05-02
  • 通讯作者: 尹卫国 E-mail:hyyinweiguo@hotmail.com
  • 基金资助:
    国家自然科学基金;国家自然科学基金;广东省科技厅课题;湖南省教育厅创新平台开放基金

Identification of in vivo?induced antigen Tp0462 of Treponema pallidum and evaluation of its application to serodiagnosis of syphilis

Liu -wen1,Deng Meixia1,zhang xiaohongzhao tie3,Luo Xi1,Zhao feijun3,Yin Weiguo   

  • Received:2017-03-27 Revised:2017-10-08 Online:2018-05-15 Published:2018-05-02
  • Contact: Yin Weiguo E-mail:hyyinweiguo@hotmail.com
  • Supported by:
    National Natural Science Foundation of China;National Natural Science Foundation of China;Guangdong Provincial Science and Technology Department Foundation;Hunan Provincial Education Department Open Innovation Platform Foundation

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摘要： 目的 筛选鉴定并评价梅毒螺旋体（Tp）体内诱生抗原Tp0462的临床血清学诊断价值。方法 提取Tp Nichols株全基因组，PCR扩增Tp0462，克隆构建重组质粒pET30a（+）?Tp0462，转化至E.coli Rosetta（DE3）菌，大量诱导表达重组蛋白Tp0462并纯化、鉴定。18只新西兰兔随机平均分为活Tp接种组、紫外线照射灭活Tp接种组和未接种对照组，接种后不同时间点抽血分离血清，用ELISA鉴定Tp0462蛋白的体内诱生性特点。ELISA法（Tp0462?ELISA）、梅毒螺旋体明胶凝集试验（TPPA）、梅毒初筛ELISA试验及快速血浆反应素环状卡片试验（RPR）试验检测336份临床梅毒患者血清样本，初步评价该蛋白的诊断价值。结果 重组质粒Tp0462?pET30a（+）在E.coli Rosetta（DE3）中最适的表达条件为0.5 mmol/L IPTG，20 ℃，180 rpm摇床诱导培养4 h。活Tp接种组血清Tp0462特异性抗体水平从第2周起呈急剧上升趋势，至5周后趋于平稳，而灭活Tp接种组及未接种对照组血清Tp0462特异性抗体水平一直处于低水平。活Tp接种组Tp0462特异性抗体水平显著高于灭活Tp接种组及未处理组（P < 0.05），而灭活Tp接种组与未处理组相比，差异无统计学意义（P = 0.256）。活Tp接种组、灭活Tp接种组Tp92抗体水平显著高于未处理组（P < 0.05），而活Tp接种组与灭活Tp接种组差异无统计学意义（P = 0.127）。与TPPA相比，Tp0462?ELISA的灵敏度和特异度分别为91.7%和98.8%，符合率为95.2%，曲线下面积为0.997。Tp0462?ELISA与梅毒初筛ELISA试验的符合率为92.3%，Kappa值为0.846；与RPR试剂盒的符合率为83.9%，Kappa值为0.293。结论 Tp0462在梅毒血清学诊断中具有较高的灵敏度和特异度，可以作为潜在的梅毒诊断候选蛋白。

关键词: 梅毒, 苍白密螺旋体, 梅毒血清诊断, 体内诱生抗原, Tp0462

Abstract: Liu Wen, Deng Meixia, Zhang Xiaohong, Zhao Tie, Luo Xi, Zhao Feijun, Yin Weiguo Institute of Pathogenic Biology, University of South China, Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, Hengyang 421001, Hunan, China （Liu W, Deng MX, Zhang XH, Zhao T, Luo X, Zhao FJ）; Clinical Laboratory, Yue Bei People′s Hospital Affiliated to Shantou University Medical College, Shaoguan 512026, Guangdong, China （Yin WG） Corresponding author: Yin Weiguo, Email: hyyinweiguo@hotmail.com 【Abstract】 Objective To screen and identify the in vivo-induced antigen Tp0462 of Treponema pallidum （Tp）, and to evaluate its value for clinical serological diagnosis of syphilis. Methods Genome-wide DNA was extracted from the Tp Nichols strain, and polymerase chain reaction （PCR） was performed to amplify the Tp0462 gene. A recombinant plasmid pET30a（+）-Tp0462 was constructed and transfected into the Escherichia coli （E.coli） Rosetta （DE3） strain. Recombinant protein Tp0462 was abundantly expressed, purified and identified. A total of 18 New Zealand rabbits were randomly and equally divided into 3 groups: viable Tp-incubating group incubated with viable Tp in the testes, inactived Tp-incubating group incubated with ultraviolet irradiation-killed Tp in the testes, and control group receiving no treatment. After incubation, blood samples were collected at different time points, and the sera were isolated for identification of characteristics of in vivo-induced antigen Tp0462. Enzyme-linked immunosorbent assay for Tp0462 （Tp0462-ELISA）, Treponema pallidum particle agglutination assay （TPPA）, preliminary syphilis screening ELISA and rapid plasma reagin （RPR） card test were applied in 336 clinical serum samples from patients with syphilis, so as to preliminarily evaluate the value of Tp0462 for the diagnosis of syphilis. Results The optimum conditions for of the recombinant plasmid Tp0462-pET30a（+） in E.coli Rosetta （DE3） strains were the treatment with 0.5 mmol/L isopropyl thiogalactoside （IPTG） on a shaker at 180 rpm for 4 hours. In the viable Tp-incubating group, the serum level of specific anti-Tp0462 antibody sharply increased from week 2, and went steady after week 5. However, the specific anti-Tp0462 antibody maintained a low level in the inactived Tp-incubating group and the control group. The viable Tp-incubating group showed a significantly higher level of specific anti-Tp0462 antibody compared with the inactived Tp-incubating group and the control group （both P < 0.05）, while no significant difference was observed between the inactived Tp-incubating group and the control group （P = 0.256）. The level of anti-Tp92 antibody was significantly higher in the viable Tp-incubating group and the inactived Tp-incubating group than in the control group （P < 0.05）, while there was no significant difference between the viable Tp-incubating group and the inactived Tp-incubating group （P = 0.127）. Compared with TPPA, the sensitivity, specificity, consistency rate and area under the curve （AUC） of Tp0462-ELISA for the diagnosis of syphilis were 91.7%, 98.8%, 95.2% and 0.997 respectively. Tp0462-ELISA was consistent to preliminary syphilis screening ELISA and RPR with a Kappa coefficient of 0.846 and 0.293, respectively. Conclusion Tp0462-ELISA has shown evidently higher sensitivity and specificity in the serodiagnosis of syphilis, and Tp0462 can serve as promising antigens for the diagnosis of syphilis.

Key words: Syphilis, Treponema pallidum, Syphilis serodiagnosis, In vivo induced antigen, Tp0462