The Detection And Construction Of Recombinant Prokaryotic Expression Plasmid Of CYP2C9Allelic Variants

ObjectiveTo establish the method for detecting CYP2C9*14, CYP2C9*25, CYP2C9*26,CYP2C9*30and CYP2C9*33alleles and to investigate the distribution frequencies of thosealleles in Guangdong popuLation. Taking CYP2C9*16as a case to explore the system forthe expression of CYP2C9allelic variants in vitro. To lay a foundation for further researchon their function, and to provide reference for the clinical reasonable application of drugsmetabolized by CYP2C9for Guangdong patients according to their genotypes.MethodACRS-PCR-RFLP genotyping assay for CYP2C9gene were performed on DNAsamples from volunteers. Possible changes on structure and physical properties of enzymesencoded by CYP2C9allelic variants were predicted by bioinformatic analysis.TheCYP2C9*16allelic variant was obtained by overlap extension PCR using wild CYP2C9gene as template and was cloned into prokaryotic expression vector pET-32a. Therecombinant expression plasmid was induced to express in vitro.ResuLts1．The frequencies of CYP2C9*14, CYP2C9*26, CYP2C9*30and CYP2C9*33were0,0.1%,0.1%and0in532DNA samples respectively, while the frequency of CYP2C9*25was0in288DNA samples. Bioinformatics analysis showed: the enzymes encoded by CYP2C9*14, CYP2C9*26, CYP2C9*30and CYP2C9*33had some changes on physicalproperties and spatial structure.indicating that the drug-metabolizing capacity of thesemutant enzymes will be affected.2． Recombinant Prokaryotic expression plasmids pET-32a-CYP2C9*1andpET-32a-CYP2C9*16were constructed successfuLly and were induced to express inEscherichia coli BL21(DE3).Conclusion1．There are almost no CYP2C9*14, CYP2C9*25, CYP2C9*33allele in GuangdongpopuLation. In the meanwhile, there are CYP2C9*26and CYP2C9*30allele in GuangdongpopuLation with a low frequency. Further research will be performed on studying thestructure and function of the corresponding enzymes.2．A prokaryotic expression system for the expression of CYP2C9allele wasestablished successfuLly, it may lay a solid foundation for further research ondrug-metabolizing kinetics of the corresponding mutant enzymes.