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  

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

作　　者：刘文[1] 邓美霞[1] 张晓红[1] 赵铁[1] 罗茜[1] 赵飞骏[1] 尹卫国 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;Clinical Laboratory, Yue Bei People＇s Hospital Affiliated to Shantou University Medical College, Shaoguan 512026, Guangdong, China)

机构地区：[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,湖南衡阳421001 [2]汕头大学医学院附属粤北人民医院检验科

出　　处：《中华皮肤科杂志》2018年第5期352-357,共6页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81373230、81301470）;广东省科技厅课题（2014A020212036）;湖南省教育厅创新平台开放基金（17K080）

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TpNichols株全基因组，PCR扩增Tp0462，克隆构建重组质粒pET30a（＋）一Tp0462，转化至EcoliR05ett0（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．coliRosetta（DE3）中最适的表达条件为0．5mmol／LIPTG，20℃，180rpm摇床诱导培养4h。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〈O．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。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 expression 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 grou

关 键 词：梅毒 苍白密螺旋体 梅毒血清诊断 体内诱生抗原 Tp0462 

分 类 号：R759.1[医药卫生—皮肤病学与性病学]