血小板生成素在枯草杆菌中的表达分泌及前肽作用

Secretion of Thrombopoietin in Bacillus subtilis and the Effect of Propeptide

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.

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. DB403CPro-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.