The Construction Of CYP3A5Gene Polymorphism Yeast Expression System And The Influence On Drug Metabolism And Effects

Objective:To construct human recombinant CYP3A5gene polymorphism brewers yeast expression system;To evaluate the differences of drug metabolism and effect by CYP3A5gene polymorphism;To explore the regulation of CYP3A5gene expression by miRNA.Methods:1.The construction and expression of CYP3A5gene polymorphism vector:CDNA fragments of YP3A5gene polymorphism was obtained by PCR, PCR products was double enzyme digested;then desired gene fragments was inserted the plasmid pYES2/CT which were double enzyme digested with the same endonuclease enzyme;then transform the e.coli JM109with CYP3A5expression vector and culture e.coli JM109with a medium containing ampicillin, screened positive clones and extracted the recombinant plasmid of CYP3A5gene polymorphism expression vector. To transform saccharomyces cerevisiae INVScl with the CYP3A5pYES2/CT recombinant expression vector, and induce the protein expression with SG culture medium, then extract the microsomal yeast protein of CYP3A5drug metabolism enzyme;2. The effect of CYP3A5gene polymorphisms on related drug metabolism:(1) CYP3A5gene polymorphism enzyme metabolized amlodipine, diltiazem, felodipine and notoginseng total saponin, and we detected the amount of drug metabolism with LC-MS and HPLC,optimized analysis with curve fitting method, calculated Km、Vmax and Vmax/Km and drawed the mie kinetics curve. (2) A total of57patients with primary hypertension who were assigned to receive amlodipine and32patients of hypertension patients after transplantation who were assigned to receive diltiazem were screened in health management center and kidney transplantation department of xiangya third hospital; Extracted peripheral blood of patients and separated plasma, On the one hand, we intervened drug in plasma with CYP3A5gene polymorphism enzyme in simulation environment in the body, and detected the blood drug concentration with LC-MS and HPLC, calculated metabolic rate of amlodipine of each group.on the other hand, we detected the polymorphism of CYP3A5, detected the blood drug concentration of each CYP3A5genetype groups with LC-MS and HPLC, respectively calculated metabolic rate.3. The effect of CYP3A5gene polymorphism on amlodipine antihypertensive effects:Screened57patients with essential hypertension in health management center of xiangya third hospital, who were assigned to receive amlodipine for4weeks, extracted DNA of patients and detected the polymorphism of CYP3A5; divided the patients into four groups According to different CYP3A5gene type:CYP3A5*1/*1group,CYP3A5*1/*3group,CYP3A5*4group and CYP3A5*6group, At the same time, measured and recorded blood pressure of the patients before and after treatment;analysed the influence of CYP3A5gene polymorphism on amlodipine antihypertensive effect.4.Study of the regulation of CYP3A5gene expression by miRNA:Targetscan, miRanda and Pictar were applied to predict the miRNA which possible effect on CYP3A5, and screened and statistics, we found that miR-410can bind with CYP3A5mRNA3’-UTR in the very great degree; In order to validate the results, we was carried out the cell transfection experiments with HepG2, the experiment is divided into5groups:blank control group,, miR-410mimic (analogue) group, miR-410mimic negative control group, miR-410inhibitor (inhibitors) group, miR-410inhibitor negative control group;The blank control group was joined the same amount of Cell culture medium, the real time and western blot were applied to detect expression1evel of CYP3A5mRNA and protein after transfection48hours.Result:1.Construction of CYP3A5gene polymorphism expression vector:for the first time to construct the recombinant cytochrome CYP3A5brewers yeast expression system in vitro, including CYP3A5*1/*1, CYP3A5*1/*3,CYP3A5*4and CYP3A5*6four single nucleotide mutation strains of saccharomyces cerevisiae expression vector.2.The determination of kinetic parameters of CYP3A5gene polymorphism enzymes metabolize related drugs:The metabolic rate of amlodipine by CYP3A5gene polymorphism: CYP3A5*1/*3>CYP3A5*6>CYP3A5wt>CYP3A5*4;The metabolic rate of diltiazem:CYP3A5wt>CYP3A5*6>CYP3A5*1/*3>CYP3A5*4; The metabolic rate of felodipine:CYP3A5wt> CYP3A5*4> CYP3A5*6>CYP3A5*3；The metabolic rate of Notoginseng total saponin:CYP3A5WT>CYP3A5*4>CYP3A5*6>CYP3A5*1/*3.The result was consistent of in vivo and in vitro.3.The determination of CYP3A5gene polymorphisms and the effect of CYP3A5genetic polymorphism on amlodipine antihypertensive effects:(1)The determination of CYP3A5gene polymorphisms:detected CYP3A5genotype of57hypertension patients who taking amlodipine in health management center of xiangya third hospital,we found a total of7CYP3A5*1/*1(12.3%),a total of28CYP3A5*1/*3(49.1%),a total of two CYP3A5*4(3.5%)and a total of twoCYP3A5*6(3.5%)；(2)the influence of CYP3A5genetic polymorphism on amlodipine antihypertensive effects:Compared with CYP3A5*1/*1genotype,the amlodipine antihypertensive effects of CYP3A5*1/*3and CYP3A5*4genotype patient significantly enhanced(P<0.05),but CYP3A5*6genotypes patient has no obvious difference(P>0.05)；4.Study of the regulation of CYP3A5gene expression by miRNA: Compared with blank control group,CYP3A5protein expression level of miR-410mimic transfection group increased significantly (P <0.01), and miR-410inhibitor transfection group contrast (P<0.001), CYP3A5protein expression level of the other two negative control groups had no significant difference. CYP3A5mRNA expression level of each groups had no significant change.Conclusion:1. For the first time to construct the recombinant human cytochrome CYP3A5brewers yeast expression system.2. CYP3A5gene polymorphism brewers yeast expression system which we had constructed can be used to evaluate the differences of clinical drug metabolism and effects.3. miR-410can regulate the expression of CYP3A5, which is a kind of transcriptional regulation mechanism.