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cDNA Cloning and Expression of Human Cytochrome P450 1A2、2C9、2E1 and 3A4

Author: XuTianXue
Tutor: YouXueFu；ChenHuiZhen；ZhangWeiXin
School: Peking Union Medical College , China
Course: Microbial and Biochemical Pharmacy
Keywords: Cytochrome P450 Drug-metabolizing enzymes Clone Heterologous expression
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 119
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Abstract


The purpose of this project is to through cloning and heterologous expression of specific gene expression recombinant cytochrome P450 drug metabolizing enzymes, and lay the foundation for the subsequent assessment of enzyme activity and enzyme metabolism studies. Build abroad receive a target gene cloning and recombinant virus containing CYP1A2, CYP2E1, CYP3A4 gene plasmid as a template for PCR amplification of the target gene, while CYP2C9 gene was amplified by PCR from a human liver cDNA. CYP1A2 and of CYP2E1, respectively, at the 5 'end add the EcoR Ⅰ digested bit points, 3' end add Kpn Ⅰ digested bit point for CYP2C9 and of CYP3A4, respectively, at the 5 'end add Sal Ⅰ digested bit point 3' end of the add Kpn Ⅰ enzyme cleavage sites. Destination cloned into pCMV-Myc vector and DNA sequencing results confirmed complete and correct target gene. After double digestion target gene fragment into by the same double digested pFastBacl transposable carrier, the recombinant plasmid was transformed into the E. coli DH10Bac competent cells generated after transposition the recombinant cosmid DNA (Bacmid,). The recombinant cosmid was transfected into Sf9 cells, which produce the recombinant baculovirus. Recombinant protein recombinant baculovirus infected Sf9 cells grow well MOI values ??of 0.01-0.1 amplified recombinant virus, the virus titer of 10 ~ 8pfu/mL or higher, then the recombinant virus at a MOI value of 5 infected Sf9 cells, expression of the target protein. Collected expressing Sf9 cells after 72h, liquid containing heavy protein dissolved by sonication, and after centrifugation. The protein concentration was measured. Western Blot analysis, recombinant protein expression analysis CYP1A2, CYP2C9 recombinant protein with sheep anti-human CYP1A2, CYP2C9 polyclonal antibody specific binding, suggesting that the correct expression of CYP1A2, CYP2C9 gene properly inserted into the expression vector. SDS-PAGE electrophoresis the results visible CYP1A2, CYP2C9 of protein reorganization CYP2E1 protein and recombinant CYP3A4 protein expression bands that CYP2E1, CYP3A4 gene is correctly expressed. In summary, the experiments using baculovirus - insect cell expression system (Bac-to-Bac baculovirus expression system) expressed CYP1A2, CYP2C9, of CYP2E1 and CYP3A4 recombinant enzyme, CYP1A2 and CYP2C9 after Western Blot Identification The results are correct. CYP2E1 and CYP3A4 After the correct protein electrophoresis test results. This experiment is to lay the foundation for the subsequent assessment of enzyme activity and enzyme metabolism studies.

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