CDNA Cloning And Expression Of Human Cytochrome P450 1A2,2C9,2E1 And 3A4

This project intends to obtain the recombinant CYP450s isoform by the method of cDNA cloning and heterologous expressing,getting ready for the next step of establishing activity of CYP450s isoform and studying of drug metabolism in vitro.1 Cloning of the target gene and construction of the recombinant virusesThe cDNA of the CYP1A2,CYP2E1,CYP3A4 gene was amplified from an unknown plasmid with CYP1A2,CYP2E1,CYP3A4 gene by PCR.The cDNA of the CYP2C9 gene was amplified from human live cDNA by PCR.EcoR I site was introduced at the 5' terminus and Kpn I sites were added to 3' terminus in the gene segment of CYP1A2 and CYP2E1.Sal I site was introduced at the 5' terminus and Kpn I sites were added to 3' terminus in the gene segment of CYP2C9 and CYP3A4.The PCR products were cloned into the pCMV-Myc vector and then were screened by restriction enzyme digestion and DNA sequencing.The results confirmed this inserted gene had the whole target gene segment.The target gene segment digested separately by EcoR I/Kpn I and Sal I/Kpn I was inserted into the vector pFastBacl which was also cleaved with two endonucleases.The harvested recombinant pFastBacl-CYP was then transformed into E.coli DH10Bac in which recombinant Bacmid would be generated by transposition. After Bacmid-CYP transfected the Sf9 cell,the recombinant viruses would be obtained.2 Expression of the recombinant proteinThe recombinant viruses were amplified with MOI 0.01—0.1 until the viral titer reached 10~8pfu/mL or higher.The recombinant viruses infected the Sf9 cells with MOI 5 to express the recombinant protein.The infected Sf9 cells were harvested after 72h. The collected cells were sonicated and the suspension of recombinant protein was obtained through standard centrifugation.The protein concentration was assayed.3 Analyzing the expression of recombinant proteinThe result of Western Blot displayed that recombinant CYP1A2 and CYP2C9 can be recognized separately by goat anti-human CYP1A2 and CYP2C9 polyclonal antibodies.It approved CYP1A2 and CYP2C9 gene had been inserted into the expression vector correctly and expressed successfully.There is separately the strip of recombinant CYP2E1 and CYP3A4 protein in the result of SDS-PAGE,which proved these two gene be expressed rightly.In conclusion,we successfully expressed the recombinant CYP 1A2,CYP2C9,CYP2E1 and CYP3A4 in baculovirus-insect cell expressing system(Bac-to-Bac expressing baculovirus system).Among them,CYP1A2 and CYP2C9 were verified by the Western Blot.CYP2E1 and CYP3A4 were proved by SDS-PAGE detecting.This project is getting ready for the next step of establishing activity of CYP450s isoform and studying of drug metabolism in vitro.