MTERF1基因真核表达载体的构建及其在C-33A细胞中的表达研究

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR OF HUMAN MTERF1 AND IT'S EXPRESSION IN C-33A CELLS

采用PCR技术从pGEM-MTERF1重组克隆载体中扩增出人MTERF1基因cDNA的开放阅读框序列,经KpnⅠ和XhoⅠ双酶切后,以pcDNA3.1(+)为真核表达载体,构建重组质粒pcDNA3.1(+)-MTERF1;通过PCR、双酶切和DNA测序方法对重组子进行鉴定;将重组真核表达质粒pcDNA3.1(+)-MTERF1转染C-33A细胞,利用免疫印迹法检测MTERF1蛋白的表达。结果显示,重组质粒pcDNA3.1(+)-MTERF1经双酶切鉴定和菌落PCR鉴定均获得大小为1 200 bp的目的条带,DNA测序结果表明该序列与GenBank中人MTERF1基因序列完全相同,插入基因的大小和方向正确;免疫印迹结果表明,MTERF1蛋白的表达水平在转染pcDNA3.1(+)-MTERF1的C-33A细胞中表达高于转染pcDNA3.1(+)的C-33A细胞。通过对人MTERF1基因的真核表达载体的构建,为进一步研究人MTERF1基因的功能奠定了基础。

The human MTERF1 open reading frame （ORF） was amplified by polymerase chain reaction （PCR） from pGEM-MTERF1 recombinant cloning vector. Selecting pcDNA3.1（＋） as eukaryotic expression vector, the recombinant products of the pcDNA3. I（＋）-MTERF 1 were gained after double digestion by restriction enzymes Kpn I and Xho I. The recombinant plasmid pcDNA3.1（＋）-MTERFI was identified by colony PCR, dual-enzyme digestion and DNA sequencing. C-33A cells were transfected with the pcDNA3.1（＋） and pcDNA3. I（＋）-MTERF 1 respectively, and the expression of MTERF2 protein was detected by Westem blotting. A specific band of 1 200 bp was detected from recombinant plasmid pcDNA3.1（＋）-MTERF1 identified by digestion of Kpn I and Xho I and PCR. DNA sequencing and identification showed that homology between this sequence and the human MTERF1 gene sequence in GenBank was 100%, and the size and the direction of the inserted gene were fight. MTERF1 protein expression in C-33A cells transfected with pcDNA3.1（＋）-MTERF1 was significantly higher than that in C-33A cells transfected with pcDNA3.1（＋）. peDNA3.1（＋）-MTERF1 eukaryotic expression vector was suecessfuUy constructed and expressed effectively in C-33A cells, which laid a foudation of further research on the function and mechanism of human MTERF1.