Published online Dec 15, 2001. doi: 10.3748/wjg.v7.i6.841
Revised: September 19, 2001
Accepted: October 20, 2001
Published online: December 15, 2001
AIM: To clone the cDNA of UGT1A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGT1A9.
METHODS: cDNA of UGT1A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E. coli DH5α. The inserted fragment, verified by DNA sequencing, was subcloned into the Hind III/Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9. CHL cells were transfected with the resultant recombinants, pREP9-UGT1A9, and selected by G418 (400 mg•L¯¹) for one month. The surviving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in medium containing G418 to obtain samples for UGT1A9 assays. The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPL C.
RESULTS: The sequence of the cDNA segment cloned, which was 1666 bp in length, was id entical to that released by GeneBank (GenBank accession number: AF056188) in co ding region. The recombinant constructed, pREP9-UGT1A9, contains the entire coding region, along with 18 bp of the 5’ and 55 bp of the 3’ untranslated region of the UGT1A9 cDNA, respectively. The cell lines established expressed the protein of UGT1A9, and the enzyme activity towards propranolol in S9 protein was found to be 101 ± 24 pmol•min-1•mg-1 protein (n = 3), but was not detectable in parental CHL cells.
CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chinese human liver and transfected into CHL cells. The CHL-UGT1A9 cell lines established efficiently expressed the protein of UGT1A9 for the further enzyme study of drug glucuronidation.