Sun Meitao, Zhang Chenggui, Mei Wen, Yang Yongqin, Yang Zefang, Zhang Xiaojuan, Xiong Wei
Pre-clinical College, Dali University, Dali 671000, China.
Yunnan Provincial Key Laboratory of Entomological Biopharmaceutical Research & Development, Dali 671000, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2017 Jun;33(6):783-788.
Objective To construct the recombinant eukaryotic expression vector of human mitochondrial transcription termination factor 2 (MTERF2) gene and determine the cellular localization by overexpressing MTERF2 in human Caski cervical cancer cells. Methods The coding sequence of MTERF2 was amplified by reverse transcription-PCR using the total RNA extracted from human cervical cancer Caski cells, and then was inserted into p3×FLAG-CMV-14 vector. After being verified by PCR and DNA sequencing, the positive recombinant plasmid was transiently transfected into Caski cells. Western blotting and immunofluorescence technique were performed to analyze the expression and distribution of human MTERF2 proteinat 24, 32 and 48 hours after transfection. Results Sequence analysis showed that the correct target gene (1158 bp) was inserted into the vector at the expected position. The target protein band was detected at M 44 000 as we had predicted in the transfected cells while not in the negative control group, which indicated MTERF2 expression vector could be successfully transfected and expressed in Caski cells. The p3×FLAG-MTERF2 protein was highly expressed and displayed a mitochondrial distribution at 24 hours post-transfection in Caski cells. Conclusion We successfully constructed the eukaryotic expression plasmid p3×FLAG-CMV-MTERF2 and expressed p3×FLAG tagged MTERF2 effectively in the mitochondria of Caski cells.
目的 构建人线粒体转录终止因子2（MTERF2）基因的重组真核表达载体，并通过在人Caski宫颈癌细胞中过表达MTERF2来确定其细胞定位。方法 以人宫颈癌Caski细胞提取的总RNA为模板，通过逆转录-PCR扩增MTERF2的编码序列，然后将其插入p3×FLAG-CMV-14载体。经PCR和DNA测序验证后，将阳性重组质粒瞬时转染至Caski细胞。在转染后24、32和48小时，采用蛋白质免疫印迹法和免疫荧光技术分析人MTERF2蛋白的表达和分布。结果 序列分析表明，正确的目的基因（1158 bp）已插入载体的预期位置。在转染细胞中检测到预期大小（M 44 000）的目的蛋白条带，而阴性对照组未检测到，这表明MTERF2表达载体可成功转染并在Caski细胞中表达。转染后24小时，p3×FLAG-MTERF2蛋白在Caski细胞中高表达并呈线粒体分布。结论 成功构建了真核表达质粒p3×FLAG-CMV-MTERF2，并使p3×FLAG标记的MTERF2在Caski细胞的线粒体中有效表达。
