Establishment of a Functional Cell Line Expressing both Subunits of H1a and H2c of Human Hepatocyte Surface Molecule ASGPR
10.1007/s11596-010-0542-1
- Author:
HU BIN
1
,
2
;
YANG YAN
;
LIU JIA
;
MA ZHIYONG
;
HUANG HONGPING
;
LIU SHENPEI
;
YU YUAN
;
HAO YOUHUA
;
WANG BAOJU
;
LU MENGJI
;
YANG DONGLIANG
Author Information
1. Division of Clinical Immunology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
2. Department of Clinical Laboratory of the First Affiliated Hospital of Xiamen University, Xiamen 361003, China
- Keywords:
ASGPR;
eukaryotic expression vector;
function cell line;
split variant
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2010;30(5):556-561
- CountryChina
- Language:Chinese
-
Abstract:
To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR Hlb) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH 1 a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector plRES2EGFP,pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRHla and H2c in 4-1-6 were confirmed by RT-PCR,Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H lb/pCDNA3.1 (neo)was transfected into cell line 4-1-6, Hlb did not down-regulate the ligand binding ability of ASGPR.The eukaryotic expression plasmid Hlb/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single Hlb nor Hlb and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both Hla and H2c stably was established. The new split variant Hlb has no effect on ASGPR binding to ASOR. ASGPRHlb alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.
