中华皮肤科杂志 ›› 2010, Vol. 43 ›› Issue (5): 332-335.

• 论著 • 上一篇    下一篇

梅毒螺旋体Tp0319重组蛋白的表达及免疫活性研究

刘双全1,汪世平2,肖勇健3,吴移谋4,赵飞俊1,曾铁兵5,张跃军6,高冬梅6   

  1. 1. 南华大学第一附属医院
    2. 中南大学湘雅基础医学院病原生物学系血吸虫病研究室
    3. 南华大学病原生物学研究所
    4. 衡阳南华大学医学院病原微生物学研究所
    5. 衡阳南华大学医学院微生物学教研室
    6.
  • 收稿日期:2009-02-24 修回日期:2009-04-23 出版日期:2010-05-15 发布日期:2012-04-12
  • 通讯作者: 刘双全 E-mail:dantelliu@163.com
  • 基金资助:

    06A061;省级基金（编号）

Expession of Tp0319 recombinant protein from Treponema pallidum and analysis of its immunocompetence

  • Received:2009-02-24 Revised:2009-04-23 Online:2010-05-15 Published:2012-04-12

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摘要：

目的 克隆和表达梅毒螺旋体Tp0319基因，并对其表达产物进行免疫活性分析。方法 挑选并克隆出Tp0319免疫优势区基因，构建原核表达载体；诱导表达并纯化重组蛋白，用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western印迹法鉴定；用纯化的重组蛋白免疫新西兰兔；间接ELISA法检测梅毒螺旋体参考血清及临床标本。结果 成功构建原核表达载体pQE32/Tp0319；高效表达和纯化出一相对分子质量约30 000的重组蛋白。用重组蛋白免疫新西兰兔，能刺激其产生高水平抗体滴度。Western印迹证明其能与梅毒患者血清发生特异性反应，阴性对照菌未见目的表达条带。间接ELISA法检测80份梅毒螺旋体参考血清（阴性、阳性各40份），阳性和阴性结果的符合率均为100%。检测200份临床梅毒血清标本及200份正常人血清，结果与梅毒螺旋体明胶凝集试验相比，灵敏度和特异度分别为92.6%和100%，符合率为96%。结论 制备的Tp0319重组蛋白具有良好的免疫活性，为进一步研究其在梅毒血清学诊断中的应用奠定一定的基础。

关键词: 免疫活性

Abstract:

Objective To clone, express Tp0319 gene from Treponema pallidum （T. pallidum）, and to assess the immunocompetence of recombinant protein. Methods The immuno-dominant region of Tp0319 gene was chosen by computer analysis, amplified from T. pallidum complete genome by PCR, subcloned into the expression vector pQE32 to construct a recombinant plasmid, pQE32/Tp0319, which was then expressed in E. coli M15. The recombinant protein was purified with Ni-NTA affinity chromatography, and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis （SDS-PAGE） and Western blot. New Zealand rabbits were immunized with the recombinant protein, and the titer of anti-Tp0319 antibodies in sera from immunized rabbits were measured with indirect ELISA. Also, indirect ELISA with the recombinant Tp0319 as coating antigen was performed to detect the anti-Tp0319 antibody in sera from 200 normal human controls and 200 patients with syphilis. Results The prokaryotic expression vector pQE32/Tp0319 was constructed successfully, and the recombinant protein Tp0319 with a molecular weight of about 30 000 was attained. Specific humoral response was elicited by the recombinant protein in New Zealand rabbits and the specific antibody titer was more than 1 ∶ 10 240 after immunization for 3 times. Western blot proved that the recombinant protein could specifically react with anti-T. pallidum IgG antibody-positive sera. Indirect ELISA was successfully developed with the recombinant Tp0319, and detected antibodies to T. pallidum in control sera with a sensitivity and specificity of 100% （40/40）, respectively. Compared with T. pallidum particle agglutination （TPPA） assay, the sensitivity and specificity of the indirect ELISA were 92.6% and 100%, respectively, in the detection of T. pallidum in sera from patients and controls, and the concordance between the indirect ELISA and TPPA was 96%. Conclusions The prepared recombinant protein shows a satisfactory immunocompetence, which may lay a foundation for its further application in the serodiagnosis of syphilis.

Key words: Immuno-competence.