Construction Of PgPXR Gene Recombinant Plasmid And Its Expression In Transfected HepG2 Cells

Objective:A recombinant plasmid pgPXR was constructed and transferred to human hepatoma HepG2 cells by using Lipofectamine TM2000 for this study. It lays foundation and provides cell models for natural active substance screening and animal physiologic and pharmacologic research in the future.Methods:RNA was extracted from fresh pig livers, reverse transcription products were used to amplify the coding sequence of pgPXR genes. After successful electrophoresis agarose gel identification, double digestion was used for the connection of PCR purified products with pCI-neo vector. The product of this connection was transferred into E.coli DH5a competent cells and was cultured in a medium with Am-picillin (Amp). Then double digestion was used for identification positive clone and the correct pCI-pgPXR were sent for sequencing. pCI-gPXR recombinant plasmid confirmed by comparison of sequencing mensuration report and the desired target sequencing. After confirmation,a large quantity of plasmids was amplified and extracted. As a result, four groups includes transfected pCI-pgPXR+pGL-3A4-Luc, transfected pCI-neo+pGL-3A4-Luc, transfected pGL-3A4-Luc and a non-transfected group were set up. They were liposome-meditated by Lipofectamine TM2000 and human hepatoma HepG2 cells were transfected with pCI-pgPXR. Five hours later, cells were treated with Rifampicin(5?M). Determination of luciferase activity assay were done after forty-eight hours, the mRNA and protein expression of pgPXR were detected by real-time fluorescent quantitative PCR and Western blotting separately in HepG2 cells.Results:(1) According to cDNA template, PCR amplified pgPXR gene coding sequence and electrophoresis agarose gel obtained a target band about 1266bp. It showed that the pgPXR encoding sequence was amplified successfully by consistent with pgPXR CDS. In addition, pgPXR recombinant plasmids were detected by electrophoresis agarose gel after transformation, culture and double digestion. There were about 1266bp pgPXR target gene band and 5400bp carrier band. The gene sequencing mensuration result of pCI-pgPXR compared with the desired target sequencing showed that both were absolutely consistent. As a result, we constructed the pCI-pgPXR recombinant plasmid successfully.(2) After five hours of liposome transfection, HepG2 cells were treated with Rifampicin(5?M) for forty-eight hours then harvest for determination of luciferase activity assay. Its result showed that the luciferase activity of transfected pCI-pgPXR+pGL-3A4-Luc group was higher than transfected pCI-neo+pGL-3A4-Luc group, transfected pGL-3A4-Luc and non-transfected group. The expression level of pgPXR mRNA and pgPXR protein in pCI-pgPXR+pGL-3A4-Luc group were also significantly higher than the other three groups. Further more, and the expression level of pgPXR did not differ between the groups of transfected pCI-neo+pGL-3A4-Luc, pGL-3A4-Luc and none-transfected.Conclusions:1. pCI-pgPXR recombinant plasmid was constructed successfully;2. Successful expression of pgPXR can be achieved by transfection of pCI-pgPXR recombinant plasmid from the human hepatoma HepG2 cells.