伍仙　何树光　刘灵芝

[摘要] 目的 分析梅毒螺旋体（Treponema pallidum，Tp）Hsp40蛋白的生物学特性并克隆其基因。 方法 通过GenBank查到Hsp40蛋白的序列，利用生物信息学软件分析其生物学特性。根据Tp Hsp40基因序列设计特异性引物，利用PCR扩增Hsp40基因，并把该DNA片段连接到pMD19-T载体，转化大肠杆菌后挑取阳性克隆采用PCR和DNA测序进行验证。 结果 Tp Hsp40基因能够编码374个氨基酸的蛋白质，其分子量为40.1 kDa，无信号肽，呈亲水性。Swissmodel 预测得到Tp Hsp40的三级结构与嗜热杆菌（Thermus thermophilus）Hsp40结构最接近。从Tp基因组中扩增得到1125 bp的Tp Hsp40基因并将其连接到pMD19-T载体。 结论 本文预测了梅毒螺旋体毒力因子Hsp40蛋白的结构和克隆得到Tp Hsp40基因。

[关键词] 梅毒螺旋体；Hsp40蛋白；生物信息学；基因克隆

[中图分类号] R377 [文献标识码] A [文章编号] 1673-9701（2018）20-0041-03

Bioinformatical analysis and gene clone of treponema pallidum Hsp40

WU Xian HE Shuguang LIU Lingzhi CHEN Chaoying ZHU Yixi MO Zuoqun

Clinical Laboratory， First Affiliated Hospital of Hunan Traditional Chinese Medical College， Zhuzhou 412000， China

[Abstract] Objective To investigate the biological characteristics of treponema pallidum （Tp） Hsp 40 and clone its gene. Methods Protein sequence of Hsp40 was found using GenBank and its biological characteristics were analyzed using bioinformatical software. Specific primer was designed based on the genetic sequence of Tp Hsp40 and PCR was used to amplify the gene of Hsp40. The DNA fragment was ligated to pMD19-T carrier and transformed to Escherichia coli. Positive clones were selected to be verified using PCR and DNA sequencing. Results Gene of Tp Hsp40 could encode a protein of 374 amino acids. The molecular weight of the protein was 40.1 kDa. The protein had no signal peptide and was hydrophilic. The tertiary structure of Tp Hsp40 predicted by Swissmodel was closest to the structure of thermus thermophilus Hsp40. Gene of Tp Hsp40 with 1125bp amplified from genome of Tp was ligated to pMD19-T carrier. Conclusion This paper predicted the structure of Hsp40， the virulence factor of Tp， and cloned the gene of Tp Hsp40.

[Key words] Treponema pallidum； Hsp40； Bioinformatics； Gene clone

梅毒螺旋體（Treponema pallidum，Tp）是引起梅毒的病原体。研究发现Tp能够感染并破坏成人多种器官，能经胎盘传入胎儿导致先天梅毒，严重者会产生死胎和流产[1，2]。目前我国梅毒患者数量呈增加趋势，仅次于病毒性肝炎和肺结核，在法定甲乙类传染病中排第三位[3-4]。如何有效预防及控制梅毒已成为一个严重的社会问题。在Tp中未发现内毒素等传统的毒力蛋白，但是通过基因工程获得Tp鞭毛蛋白、铁蛋白TpF1、外膜蛋白和趋化蛋白在体外能够诱导细胞产生炎症反应，提示这些蛋白可能是Tp致病中的关键蛋白[5-9]。寻找Tp新的毒力蛋白是研究其致病机制的关键[9]。热休克蛋白（heat shock protein，Hsp）是一组具有保守结构域的蛋白超家族。在细菌受到应激和胁迫时大量表达，帮助细菌适应环境。最近发现Hsp40蛋白在细菌致病过程中可能发挥重要功能[10-13]。……