Zhang Yong-li, Yu Xin-bing, Wu De, Wu Zhong-dao, Bi Hui-xiang
Department of Parasitology, Sun Yat-Sen University of Medical Sciences, Guangzhou 510089, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2005 Feb 28;23(1):18-23.
To construct prokaryotic recombinant plasmids of transcriptional co-activator (TC) gene of Clonorchis sinensis, express and purify the recombinant protein and analyze its biological function.
A pair of primers was designed according to the known sequence of TC gene. The TC gene fragment was amplified by PCR. After purification and digestion with BamH I and Sal I, the TC gene was connected to the prokaryotic expression vectors, pGEX-4T-1 and pET30a(+). By cloning target gene into these vectors, pGEX-4T-1 and pET30a(+), prokaryotic recombinant plasmids of TC gene were constructed and transferred into E. coli BL21. The positive expressed recombinants were detected by SDS-PAGE and Western blotting. Immobilized metal (Ni2+) chelation affinity chromatography was used to purify His-TC produced by the expression of the recombinant protein pET30a(+)-TC.
The recombinant plasmids, pGEX-4T-1-TC and pET30a(+)-TC, were constructed successfully. SDS-PAGE testified that the molecular weight of the recombinant protein was correct. Western blot analysis of GST-TC recombinant protein testified that the recombinant protein could be recognized by immunized rabbit serum, which means the protein is GST-immune active and the clone can express recombinant Clonorchis sinensis antigen. After affinity chromatography of the pET-TC protein, there was only one protein band with expected size on the SDS-PAGE gel.
The TC gene was screened from cDNA library of adult Clonorchis sinensis, cloned, expressed and purified. The purified protein of TC gene will be of importance for further research on the biological function of the gene.
构建华支睾吸虫转录共激活因子（TC）基因的原核重组质粒，表达并纯化重组蛋白，分析其生物学功能。
根据TC基因已知序列设计一对引物，通过PCR扩增TC基因片段。经纯化及BamH I和Sal I双酶切后，将TC基因连接到原核表达载体pGEX-4T-1和pET30a(+)中。通过将目的基因克隆到这些载体中构建TC基因的原核重组质粒，并将其转入大肠杆菌BL21。采用SDS-PAGE和Western印迹法检测阳性表达重组子。利用固定化金属（Ni2+）螯合亲和层析法纯化重组蛋白pET30a(+)-TC表达产生的His-TC。
成功构建了重组质粒pGEX-4T-1-TC和pET30a(+)-TC。SDS-PAGE证实重组蛋白分子量正确。对GST-TC重组蛋白进行Western印迹分析表明，该重组蛋白能被免疫兔血清识别，这意味着该蛋白具有GST免疫活性，且该克隆可表达重组华支睾吸虫抗原。对pET-TC蛋白进行亲和层析后，SDS-PAGE凝胶上仅出现一条预期大小的蛋白条带。
从华支睾吸虫成虫cDNA文库中筛选出TC基因，进行克隆、表达及纯化。纯化的TC基因蛋白对该基因生物学功能的进一步研究具有重要意义。
