中华皮肤科杂志 ›› 2010, Vol. 43 ›› Issue (7): 489-492.

• 论著 • 上一篇    下一篇

梅毒螺旋体Tp0821基因的克隆、表达、纯化及免疫活性研究

伍宁1,肖勇健1,顾伟鸣2,刘双全3,赵飞俊3,张跃军4,吴移谋5   

  1. 1. 南华大学病原生物学研究所
    2. 上海市皮肤病性病医院
    3. 南华大学第一附属医院
    4.
    5. 衡阳南华大学医学院病原微生物学研究所
  • 收稿日期:2009-09-30 修回日期:2009-11-10 出版日期:2010-07-15 发布日期:2010-07-13
  • 通讯作者: 伍宁 E-mail:wuning0204@yahoo.com.cn
  • 基金资助:

    湖南省教育厅重点资助项目;湖南省自然科学基金课题

Gene cloning, expression and purification of Tp0821, a membrane lipoprotein of Treponema pallidum and its immunocompetence

  • Received:2009-09-30 Revised:2009-11-10 Online:2010-07-15 Published:2010-07-13

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摘要：

目的 构建梅毒螺旋体（Tp）膜脂蛋白Tp0821的重组质粒，表达、纯化其相应蛋白，研究其免疫活性。方法 构建重组质粒pQE32/Tp0821，诱导表达其相应蛋白，以纯化的重组蛋白免疫新西兰兔，制备多克隆抗体并测定其效价。建立间接ELISA法，检测80份梅毒参比血清、临床经FTA-ABS确诊的阳性血清各150份。结果 成功构建重组质粒pQE32/Tp0821，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析重组蛋白分子量与预期结果相符。将亲和层析后所获高纯度重组蛋白免疫新西兰兔，制备的多克隆抗体效价达1 ∶ 6400。间接ELISA法检测梅毒参比血清、临床梅毒患者血清，与间接免疫荧光螺旋体抗体吸附试验（FTA-ABS）法比较，其灵敏度、特异度分别为92.6%和98.6%，差异有统计学意义（P ＜ 0.05）。结论 Tp0821重组蛋白具有较好的免疫活性，可用于梅毒血清学检测。

关键词: 梅毒螺旋体, Tp0821重组蛋白, 免疫活性

Abstract:

Objective To construct a recombinant plasmid encoding Tp0821, a membrane lipoprotein of T. pallidum, express and purify this protein, and to evaluate its immunocompetence. Methods The recombinant plasmid pQE32/Tp0821 was constructed and induced to express the corresponding protein. Then, New Zealand rabbits were immunized with purified recombinant protein to prepare polyclonal antibodies, and the titer of polyclonal antibody was determinated. Indirect ELISA was developed with the recombinant protein of T. pallidum as coating antigen to detect 80 control sera and 150 FTA-ABS-positive sera. Results The recombinant plasmid pQE32/Tp0821 was constructed and a fusion protein with expected molecular weight was expressed. Specific humoral response was elicited by the recombinant protein in New Zealand rabbits and the antibody titer reached 1 ∶ 6 400. Compared with FTA-ABS test, the indirect ELISA showed a sensitivity and specificity of 92.6% and 98.6%, respectively, in the detection of control and clinical sera. Conclusion The recombinant protein Tp0821 shows excellent immunocompetence, which can be applied to the serological diagnosis of syphilis.

Key words: Treponema pallidum, Tp0821 recombinant protein, Immuno-competence.