本地毛形线虫49kuES蛋白结构基因在昆虫细胞中的表达

Expression of 49 ku ES structural protein gene from Trichinella nativa in insect cells

根据BAC-TO-BAC杆状病毒表达系统的供体质粒,设计表达引物,从克隆质粒上扩增本地毛形线虫(Trichinella nativa)49kuES结构蛋白基因TNPG,酶切后与供体质粒连接,在大肠杆菌中克隆,将克隆有外源基因的供体质粒转化DH10Bac感受态菌,该菌含有杆状病毒穿梭载体,供体质粒中的外源基因在辅助质粒编码的转座酶作用下,通过转座而插入杆状病毒基因组中,筛选含有重组质粒的Bacmid DNA,用脂质体介导法转染昆虫细胞获得重组病毒。SDS-PAGE电泳显示表达蛋白为36ku的融合蛋白,Western blot检测表明表达的融合蛋白能够被小鼠旋毛虫的阳性血清识别。实验结果表明,本地毛形线虫的ES融合蛋白的制备将在诊断方法的建立和免疫研究方面具有很好的应用前景。

A pair of primers was designed according to the sequence of donor plasmid pFastBAC HTa （BAC-TO-BAC Baculovirus Express System）. The target gene was linked with donor plasmid and the recombinant plasrnid was transformed into DH10Bac. The bacmid plasmid DNA was transfected insect cells through lipid-mediated transfection. SDS-PAGE and Western blot analysis showed that expressed target protein has a MW 36 ku and could be specifically reacted to positive serum of mouse against Trichinella nativa.