论文部分内容阅读
目的构建人线粒体转录终止因子2(MTERF2)基因真核表达载体,并在人Caski宫颈癌细胞中过表达,观察其编码蛋白质在Caski细胞中的定位。方法从Caski细胞中提取总RNA,采用反转录PCR扩增人MTERF2基因可读框序列,将其重组于p3×FLAG-CMV-14载体中,利用PCR和DNA测序鉴定重组子正确性;并用脂质体法转染至Caski细胞,转染24、32、48 h,采用Western blot法检测MTERF2蛋白水平,通过免疫荧光细胞化学染色观察其亚细胞定位情况。结果经过PCR和DNA测序鉴定,人MTERF2基因可读框正确地插入到真核表达质粒中,大小为1158 bp,表达的蛋白相对分子质量(Mr)为44 000,与预计大小相符。转染p3×FLAG-MTERF2质粒的Caski细胞培养24 h,可检测到目的蛋白表达,该蛋白主要定位于线粒体中。结论在Caski细胞成功表达了MTERF2蛋白并证实其定位于线粒体。
Objective To construct an eukaryotic expression vector of human telomerase reverse transcription - terminator 2 (MTERF2) gene and overexpress it in human Caski cervical carcinoma cells. Observe the localization of the encoded protein in Caski cells. Methods The total RNA was extracted from Caski cells. The open reading frame of human MTERF2 gene was amplified by reverse transcription PCR. The recombinant plasmid was then recombined in p3 × FLAG-CMV-14 vector. The correctness of recombinant plasmid was confirmed by PCR and DNA sequencing. The transfected cells were transfected with Caski cells by Lipofectamine 2000 for 24,32 and 48 h. The protein level of MTERF2 was detected by Western blot and subcellular localization was observed by immunofluorescence staining. Results After PCR and DNA sequencing, the human MTERF2 gene was inserted into the eukaryotic expression plasmid correctly. The size of the human MTERF2 gene was 1158 bp. The relative molecular mass of the expressed protein was 44 000, which was consistent with the expected size. Caski cells transfected with p3 × FLAG-MTERF2 plasmid were cultured for 24 h, and the expression of the target protein was detected. The protein mainly located in the mitochondria. Conclusion The MTERF2 protein was successfully expressed in Caski cells and confirmed its localization in mitochondria.