[Construction, identification and expression of recombinant eukaryotic vector pEGFP-BMI-1].
This study was purposed to construct and identify the mammalian expression vector of pEGFP-BMI-1 and to detect whether it could express in human cervix cancer cell line HeLa. The cDNA fragment of BMI-1 obtained by RT-PCR was inserted into pEGFP-N1. The recombinant plasmid was confirmed by restriction enzyme digestion, PCR and DNA sequencing. pEGFP-BMI-1 was transfected into HeLa cells with lipofectamine 2000. The expression of pEGFP-BMI-1 was determined by EGFP fluorescence and Western blot analysis. SYBR Green I real-time RT-PCR was used to quantitate P16INK4a mRNA. The results showed that the correct construction of the recombinant plasmid pEGFP-BMI-1 has been shown by restriction enzyme digestion, PCR and DNA sequencing. pEGFP-BMI-1 could express BMI-1-EGFP fusion protein in HeLa cells. Real-time RT-PCR showed that P16INK4a mRNA expression was reduced to 9.2%. It is concluded that the vector of pEGFP-BMI-1 has been successfully constructed and it can be expressed in HeLa cells. This work has laid foundations for further study on biological functions and potential application of BMI-1.
Chen
Feng-Hua
FH
Department of Laboratory Examination, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Hu
Li-Hua
LH
Wang
Lin
L
Li
Yi-Rong
YR
D001483:Base Sequence; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
C069077:BMI1 protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D063150:Polycomb Repressive Complex 1