Construction, identification and expression of recombinant eukaryotic vector pEGFP-BMI-1 / 中国实验血液学杂志

ABSTRACT

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.