华东师范大学学报(自然科学版) ›› 2005, Vol. 2005 ›› Issue (1): 117-121.

• 生命科学 环境科学 • 上一篇    下一篇

血小板生成素在枯草杆菌中的表达分泌及前肽作用

张美华,周建,杨宇峰,吴自荣   

  1. 华东师范大学 生命科学学院, 上海200062
  • 收稿日期:2003-11-12 修回日期:2003-12-21 出版日期:2005-03-25 发布日期:2005-03-25
  • 通讯作者: 吴自荣

Secretion of Thrombopoietin in Bacillus subtilis and the Effect of Propeptide(Chinese)

ZHANG Mei-hua,ZHOU Jian,YANG Yu-feng,WU Zi-rong   

  1. School of Life Science,East China Normal University,Shanghai200062,China
  • Received:2003-11-12 Revised:2003-12-21 Online:2005-03-25 Published:2005-03-25
  • Contact: WU Zi-rong

摘要： 利用PCR方法扩增得到枯草杆菌本身的表达调控序列SP（含有信号肽）和Pro序列（包含信号肽和前肽），分别与血小板生成素(TPO)结构基因及枯草杆菌载体片段连接，构建两个重组TPO枯草杆菌表达载体，转化受体菌，得到DB403（SP-TPO）和DB403（Pro-TPO）重组克隆，比较有无前肽对其表达分泌的作用.用ELISA和Western Blot检测TPO的表达分泌，发现DB403（Pro-TPO）能够表达分泌TPO，而DB403（SP-TPO）未发现TPO的分泌.对小鼠腹腔注射DB403（Pro-TPO）发酵浓缩液，发现能够明显增加血小板数目，与自身对照相比增加84.2％，与生理盐水对照比较增加99.7％.以上实验说明Pro-TPO能够表达分泌有明显活性的TPO蛋白，而且前肽对外源蛋白的分泌是必要的.

关键词: 血小板生成素, 枯草杆菌, 分泌, 前肽, 血小板生成素, 枯草杆菌, 分泌, 前肽

Abstract: To understand the secretory expression of heterologous protein in Bacillus subtilis, especially of those from eucaryotes, also to reveal the role of propeptide, two recombinant plasmids containing TPO gene were cloned in Bacillus subtilis with or without propeptide, named Pro-TPO, SP-TPO respectively. PCR production fragment SP containing signal peptide was ligated with TPO and vector. The recombinant plasmid was transformed into DB403 and a positive clone DB403(SP-TPO) was obtained. Another recombinant was available at the same time with the regulatory sequence Pro containing signal peptide and propeptide, named DB403(Pro-TPO). The secretion of TPO of the three recombinants can be valued by means of ELISA and Western blot. DB403(Pro-TPO) was shown to have the ability of secreting TPO, however DB403(SP-TPO) was found no secretion. DB403(Pro-TPO) was administrated peritoneally to Babl/c mice and got the number of platelet increased by 84.2%(self control) and 99.7%(saline control). The results implied that TPO is expressed with native activity and propeptide is essential for the secretion of TPO.

Key words: Bacills subtilis, secretion, propeptide, thrombopoietin, Bacills subtilis, secretion, propeptide

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