Construction of cytochrome P450 2C9 * 8 and CYP2C9 * 27 lentiviral expression plasmid and stable express in HEK293 cells
10.7619/jcmp.201807001
- VernacularTitle:细胞色素P4502C9*8与CYP2C9*27慢病毒表达载体的构建及在HEK239T细胞中的稳定表达
- Author:
Kai LIAO
1
;
Yong LIU
;
Zijin WAN
;
Wei LI
Author Information
1. 扬州大学医学院
- Keywords:
cytochrome P4502C9 (CYP2C9);
CYP2C9 * 8;
CYP2C9 * 27;
lentivirus;
HEK293T
- From:
Journal of Clinical Medicine in Practice
2018;22(7):1-6
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct lentiviral expression plasmids of CYP2C9,CYP2C9 * 8 and CYP2C9 * 27,and to express CYP2C9,CYP2C9 * 8 and CYP2C9 * 27 stably in HEK2C9T cells.Methods CYP2C9 coding region was obtained by reverse transcription PCR from the human liver total RNA,and then cloned into the mammalian expression vector MigR1.CYP2C9 * 8 or CYP2C9 * 27 coding sequences were obtained from MigR1-CYP2C9 by overlapping PCR and then cloned into MigR1.The coding sequences were introduced to HEK293 cells by lentivirus,and 293T-2C9,293T-2C9 * 8 and 293T-2C9 * 27 were screened by flow cytometry and monoclonal picking.Cellular fluorescence,qRT-PCR,Western blot and the metabolic activity assays were used to identify the cells.Results MigR1-CYP2C9,MigR1-CYP2C9 * 8 and MigR1-CYP2C9 * 2293T-2C9 * 8 plasmids were constructed.All monoclonal cells 293T-2C9,293T-2C9 * 8 and 293T-2C9 * 27 cell lines expressed GFP after 30 passages.Both qRT-PCR and Western blot assay showed CYP2C9 expression in the cells.Microsomes of the three cell lines were capable of catalyzing diclofenac to 4-OH diclofenac.Conclusion 293T-2C9,293T-2C9 * 8 and293T-2C9 * 27 celllines stably expresse CYP2 C9,CYP2C9 * 8 and CYP2C9 * 27,respectively.The cell lines provide useful tools for the function investigation of CYP2C9 * 8 and CYP2C9 * 27.
