日本血吸虫翻译控制肿瘤蛋白基因的杆状病毒表达载体构建和在昆虫细胞中的表达

目的构建日本血吸虫的翻译控制肿瘤蛋白(SjTCTP)基因的杆状病毒重组供体质粒pFASTA-TCTP,用该重组转移质粒转化含有杆状病毒表达载体以构建BacmidGFP-TCTP,感染昆虫细胞进行表达。方法将已克隆的SjTCTP基因与线性化的pFASTA进行连接为pFASTA-TCTP,使pFASTA-TCTP与DH10BAC感受菌进行转座重组,利用抗性及蓝白斑筛选重组BacmidGFP-TCTP克隆,提取BacmidGFP-TCTP转染昆虫细胞Sf9获得有感染力的病毒,利用病毒感染Sf9细胞进行蛋白表达,通过荧光镜观察、RT-PCR、SDS-PAGE和Western-Blotting检测表达情况。结果获得了重组SjTCTP的杆状病毒,细胞能表达出与SjTCTP单抗结合的、分子量为23kDa左右的融合有组氨酸标签的目的蛋白。结论SjTCTP能在昆虫细胞中获得良好表达,为其的分子生物学活性研究奠定了基础。

Construction and expression of the Baculovirus expression vector for translationally controlled tumor protein gene of Schistosoma japonicum

To construct the Baculovirus expression vector for translationally controlled tumor protein (TCTP) gene of Schistosoma japonicum and to express in insect sf9 cell line, the TCTP eDNA was cloned into plasmid pFASTA, and therecombinant plasmid was transformed to competent cells DH10Bat.A transformant containing the target gene was obtained and named as BacmidGFP-SjTCTP. After transfecting this transformant into Sf9 cells, its expression in Sf9 cells was detected by fluorescent microscopic examination, RT-PCR, SDS-PAGE and Western blotting. The experimental results showed that the recombinant buculovirus was obtained and the transformant BacmidGFP-SjTCTP was expressed in insect Sf9 cells. This recombinant transformant can be used as the protein accessed high biological characteristics for the functional study on TCTP of S.japonicum.