[Molecular cloning, tissue distribution and expression in engineered cells of human orphan receptor GPR81].
The gpr81 was amplified by polymerase chain reaction (PCR) using human fetus kidney cDNA and whole blood genome DNA as template, respectively. The expression profile of gpr81 in human fetus was analyzed by RT-PCR and the result indicated GPR81 mRNA was most abundant in fetus liver and heart. In addition, the deduced amino acid of GPR81 was compared with other related molecules by Clustal w/x software, and a molecular phylogenetic tree was constructed with Treeview software. It was showed that GPR81 had the highest homology with nicotinic acid receptor in amino acids. After sequence identification, gpr81 was inserted into the plasmid pcDNA3. 1 (-)/his-mycA and then transfected into Chinese hamster ovary cell (CHO-K1). With the selection of G418, an engineered cell line which could stably express gpr81 was obtained by the indication of RT-PCR and Western-blot detection. The establishment of the cell line will serve as means for further study of GPR81.
Wu
Fang-Ming
FM
Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China.
Huang
Huo-Gao
HG
Hu
Ming
M
Gao
Yue
Y
Liu
Yong-Xue
YX
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000235:genetics; D005333:Fetus / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection
D018076:DNA, Complementary; C533610:HCAR1 protein, human; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins