从肝脏组织中克隆UGT1A9 cDNA及其在CHL细胞中的表达。

Cloning of UGT1A9 cDNA from liver tissues and its expression in CHL cells.

作者信息

Li X, Yu Y N, Zhu G J, Qian Y L

机构信息

Department of Pathophysiology, School of Medicine, Zhejiang University, Hangzhou, China.

出版信息

World J Gastroenterol. 2001 Dec;7(6):841-5. doi: 10.3748/wjg.v7.i6.841.

DOI:10.3748/wjg.v7.i6.841

PMID:11854913

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4695606/

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 UGT1 A9 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 DH5(alpha). 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 x 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 forUGT1A9 assays. The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPLC.

RESULTS

The sequence of the cDNA segment cloned, which was 1666 bp in length, was identical to that released by Gene Bank (GenBank accession number: AF056188) in coding 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 theUGT1A9 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 x min(-1) x 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-UGT1 A9 cell lines established efficiently expressed the protein ofUGT1A9 for the further enzyme study of drug glucuronidation.

摘要

目的

从中国正常人肝脏中克隆尿苷二磷酸葡萄糖醛酸基转移酶1A9（UGT1A9）的互补DNA（cDNA），并建立表达人UGT1A9的中国仓鼠肺（CHL）细胞系。

方法

采用逆转录-聚合酶链反应从信使核糖核酸（mRNA）转录UGT1A9的cDNA，并克隆至pGEM-T载体，在宿主菌大肠杆菌DH5α中扩增。经DNA测序验证插入片段后，亚克隆至哺乳动物表达载体pREP9的Hind III /Not I位点，构建质粒pREP9-UGT1A9。用所得重组体pREP9-UGT1A9转染CHL细胞，并用400 mg/L的G418筛选1个月。收获存活的克隆（CHL-UGT1A9）作为一个群体，在含G418的培养基中传代培养以获得用于UGT1A9检测的样本。通过高效液相色谱法测定CHL-UGT1A9对细胞S9蛋白中普萘洛尔的酶活性。

结果

克隆的cDNA片段长度为1666 bp，其编码区序列与基因库（GenBank登录号：AF056188）公布的序列一致。构建的重组体pREP9-UGT1A9分别包含UGT1A9 cDNA完整的编码区，以及5′端18 bp和3′端非翻译区55 bp。所建立的细胞系表达UGT1A9蛋白，其对S9蛋白中普萘洛尔的酶活性为101±24 pmol·min-1·mg-1蛋白（n = 3），而亲代CHL细胞中未检测到该活性。