Original Research
Copyright ©The Author(s) 2001. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 15, 2001; 7(6): 841-845
Published online Dec 15, 2001. doi: 10.3748/wjg.v7.i6.841
Cloning of UGT1A9 cDNA from liver tissues and its expression in CHL cells
Xin Li, Ying-Nian Yu, Ge-Jian Zhu, Yu-Li Qian
Xin Li, Ying-Nian Yu, Ge-Jian Zhu, Yu-Li Qian, Department of Pathophysiology, School of Medicine, Zhejiang University, Hangzhou 310006, Zhejiang Province, China
Xin Li, College of Pharmcy, Zhejiang University, Hangzhou 310006, Zhejiang Province, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China (C39370805), Zhejiang Provincial Natural Science Foundation (300487) and the Excellent Youth Scientist Fund of Zhejiang Province
Correspondence to: Prof. Ying-Nian Yu, Department of Pathophysiology, School of Medicine, Zhejiang University, Hangzhou 310006, Zhejiang Province, China. zjhsin@yahoo.com
Telephone: +86-571-87217203, Fax: +86-571-87217149
Received: June 19, 2001
Revised: September 19, 2001
Accepted: October 20, 2001
Published online: December 15, 2001
Abstract

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.

Keywords: UGT1A9; cloning; glucuronidation; cell lines