UGT1A1Gene Recombinant Plasmid Construction And Identification

ObjectiveThe recombinant plasmids of UGT1A1genotypes of wild type and G71Rhomozygous mutation were respectively constructed and then transfected toCOS-7cells by using lipofectamine for detecting the expression of UGT1A1.The study in this dissertation lays the foundation for the resrarch of UGT1A1gene mutations expression in vitro.Methods（1） The target genes of UGT1A1wild-type and G71R （211G�'A）homozygous mutant were cloned respectively by using the gene recombinationtechnology. The target genes were connected with pIRES2-EGFP carrierplasmid after double enzyme digestion, and then the productions weretransformed to the e.coli DH5α. The recombinant plasmids were analysised byPCR and gene sequencing to determine whether the construction of recombinantplasmids was successful.（2） After setting the transfected target gene group, the transfected emptygroup vector group, and the non-transfected group, the recombinantplasmids and empty plasmids were respectively transfected to COS-7cellsusing lipofectamine, and then observed the transfection rate under thefluorescence microscope.（3） The real-time fluorescent quantitative PCR （RT-PCR） and Westernblotting technique （WB） methods were used to detect UGT1A1mRNA andprotein expression in COS-7cells.Results（1） About1622bp size stripes were obtained in agarose gel electrophoresiswhile UGT1A1wild type and G71R homozygous mutation genes wereamplificated by PCR.（2） The positive recombinant plasmids were analyzed by gene sequencingand sequence alignment. The results showed that UGT1A1wild typerecombinant plasmid had the same base sequence completely with UGT1A1mRNA （NM₀00463）, and the homology was100%, while the differencebetween G71R homozygous mutation type recombinant plasmid sequence andUGT1A1mRNA （NM₀00463） was just a base （211G�'A）, with the expectedresults point mutations and the homology was99%.（3） Under the fluorescent microscop, significant green fluorescent tags hadbeen seen in COS-7cells which transfected by recombinant plasmids and emptyplasmids for48h，and the transfection efficiency was about40%. The greenfluorescent tags did not seen in notransfection group.（4） The UGT1A1mRNA and protein expression level in COS-7cells ofwild type and mutant groups were significantly higher than the empty plasmidstransfection groups and not transfection groups （P <0.05）. No significant differences between the empty plasmids transfection groups and not transfectiongroups （P>0.05）.Conclusion（1） The recombinant plasmids of UGT1A1wild type and G71Rhomozygous mutation type were constructed successfully.（2） The COS-7cells transfected by recombinant plasmids of UGT1A1wildtype and G71R homozygous mutation type could let UGT1A1be successfullyexpressed.