Expression And Function Of UGT1A1 Gene Wild Type And Q239X Mutant In Cos-7 Cells

Objective To investigate the expression and function of the uridine diphosphate glucuronyltransferase 1A1(UGT1A1)gene wild type and Q239X mutant in African green monkey kidney cells(cos-7 cells).Methods The UGT1A1 gene wild type overexpression,UGT1A1 gene Q239X mutant overexpression lentiviral vector and negative lentiviral vector were constructed,and the cos-7 cells cultured in vitro were transfected.After 72 hours of transfection,the transfection efficiency of cos-7 cells was observed by fluorescence inverted phase contrast microscope and flow cytometry.The cells were stably transfected with 1.0 mg/L puromycin for 1 week.The cos-7 cell line stably transfected with UGT1A1 gene was identified by DNA sequencing.Cos-7 cells were divided into 5 groups:blank control group,negative lentivirus group,UGT1A1 gene wild type group,UGT1A1 gene Q239X heterozygous mutant group,UGT1A1 gene Q239X Homozygous mutant group.The DNA,mRNA and protein expression of UGT1A1 gene in cos-7 cells were detected by PCR,Real-Time PCR and Western Blot.Standard curve of unconjugated bilirubin was drawn.Design two groups of reactions,wild type is the control group,Q239X heterozygous mutant is the experimental group,catalyzing unconjugated bilirubin and uridine diphosphate glucuronic acid(UDPGA)begin the binding reaction,high performance liquid chromatograph(HPLC)quantitative detection of unbound bilirubin concentration changes,analysis of the catalytic activity of two UGT1A1 enzymes on unbound bilirubin.Results 1.Fluorescence inverted phase contrast microscopy showed that the negative virus group,UGT1A1 gene wild type group,UGT1A1 gene Q239X heterozygous mutation group,UGT1A1 gene Q239X homozygous mutation group cos-7 cells showed obvious green fluorescence expression and cell state was good,flow The cytometry detection transfection efficiency is>85%,and the transfection efficiency is high,which can be used in subsequent experiments.2.DNA sequencing results showed that the stably overexpressing UGT1A1 gene wild type,Q239X heterozygous mutant,and Q239X homozygous mutant cos-7 cell line were successfully constructed.3.The primers were designed and amplified by PCR,and the amplified products were identified by agarose gel electrophoresis.The results showed that the UGT1A1 gene wild type,Q239X heterozygous mutant,and Q239X homozygous mutant cos-7 cells showed UGT1A1 in DNA.Gene expression,while the blank control group and the negative lentivirus group had no UGT1A1 gene expression.4.The results of Real-Time PCR showed that the mRNA expression of UGTIA1 gene in wild type,Q239X heterozygous mutant and Q239X homozygous mutant cos-7 cell of UGT1A1 gene was significantly higher than that of blank control group and negative lentivirus group.The difference was statistically significant.Significance(P<0.05).However,there was no significant difference in the mRNA expression of UGT1A1 gene wild type,Q239X heterozygous mutant and Q239X homozygous mutant UGT1A1 gene(P>0.05).5.Western blot analysis showed that UGT1A1 gene wild type,Q239X heterozygous mutant cos-7 cells showed UGT1A1 protein expression at 55kD,protein expression was different(P<0.05),while blank control group,negative lentivirus group,Q239X homozygous The mutant group had no UGT1A1 protein expression.6.Draw a standard curve of unbound bilirubin and obtain the equation:y=68028x+6813.7,R2=0.9923(where x is the unconjugated bilirubin concentration?M,y is Unbound bilirubin peak area),the unbound bilirubin has a good linear relationship with the unbound bilirubin peak area in the concentration range of 0.25-2?M.7.UGT1A1 wild type,Q239X heterozygous mutant cos-7 cell model were used to catalyze the glucuronidation of unconjugated bilirubin and UDPGA,The UGT1A1 wild-type and Q239X heterozygous mutants have the strongest catalytic activity within 15 min,resulting in the largest amount of bound bilirubin production,the largest decrease in unbound bilirubin concentration,and the Q239X heterozygous mutant enzyme catalyzes the decrease of unbound bilirubin concentration lower than UGT1A1 wild enzyme.Conclusion 1.The UGT1A1 gene Q239X mutation does not affect the transcriptional expression of the gene.2.The UGT1A1 gene Q239X homozygous mutation makes its translated glutamine into a stop codon,gene expression is terminated,and no UGT1A1 protein is produced.3.The UGT1A1 gene Q239X heterozygous mutation can express UGT1A1 protein,and the UGT1A1 enzyme produced by it can catalyze the unconjugated bilirubin ability lower than the UGT1A1 gene wild type.