Construction of stable expression of human thyrotropin receptor alpha-subunits on HEK 293T cells.
- Author:
Gongshun TANG
- Publication Type:Journal Article
- MeSH:
Genetic Vectors;
HEK293 Cells;
Humans;
Lentivirus;
Plasmids;
Receptors, Thyrotropin;
biosynthesis;
Transfection
- From:
Journal of Biomedical Engineering
2014;31(1):146-151
- CountryChina
- Language:Chinese
-
Abstract:
The aim of this study was to establish stable expression of human thyroid stimulating hormone receptor (TSHR) alpha-subunit (hTSHRA) on human embryonic kidney 293T (HEK 293T). HEK 293T cell lines with stable expression of hTSHRA could be used for detecting affinity between hTSHRA and potential TSHR blocking-peptide. We firstly constructed hTSHRA gene into lentiviral vectors GV218. The sequence comparison indicated that we had constructed GV218-hTSHRAA. Western blot demonstrated the 52 kD aim band of hTHSRA on over-expressed HEK 293T cells. GV218-hTSHRA constructions and pHelper were then co-transfected into HEK 293T cells to form packaging plasmid. The HEK 293T cells that stably expressed hTSHRA could also express green fluorescent pro tein. The titer of lentiviral packaging vector is 2 x 10(8) TU/mL with qPCR. The lentiviral packaging vector thereafter was transfected into HEK 293T cells again. The hTSHRA expressed on the HEK 293T cells. Human TSHRA stab ly expressed on HEK 293T upon continuously passaging. Therefore, we established hTSHRA stable expression on HEK 293T cells by constructing GV218-hTHSR lentiviral packaging vector. It is a useful tool for studying TSHR affinity with anti-thyroid peptide.
