Establishment of CHO cell line expressing human MCHR2 gene and research of its molecular characteristics.
The whole length of MCHR2 gene cDNA fragment was amplified by PCR using human fetal brain cDNA library as template. The pcDNA3.1 (+)/MCHR2 eukaryotic expression vector was constructed successfully. The recombinant pcDNA3.1 (+)/MCHR2 plasmid was transfected into Chinese hamster ovary (CHO) cell by lipofectamine 2000, after G418 selection and then the CHO cell line expressing MCHR2 gene was established. The MCHR2 gene expression was tested by RT-PCR, western blotting and immunofluorescence. The maximum binding (B(max)) of CHO cell line was 309.97 +/-1.14 fM x mg(-1) protein and the dissociation constant (K(d) value) was 0.170 +/- 0.0006 nM. MCH could stimulate Ca2+ release, its 50% effective concentration (EC50) was 2.32 +/- 0.01 nM. The construction of the CHO cell line and the research of MCHR2 molecular characteristics have established a good experimental basis for the further research about the function of MCHR2 gene.
Yuan
Chengfu
C
Department of Biochemistry & Molecular Biology, Molecular Medicine & Cancer Research Center, Chongqing Medical University, YiXueYuan Road No. 1, Chongqing 400016, China.
Bu
Youquan
Y
Sengupta
Joyeeta
J
Yang
Junxia
J
Huang
Xiuning
X
Cheng
Li
L
Zhang
Qin
Q
Yi
Faping
F
Liu
Geli
G
Song
Fangzhou
F
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D006801:Humans; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*; D011979:Receptors, Pituitary Hormone / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; C431280:MCHR2 protein, human; D043562:Receptors, G-Protein-Coupled; D011979:Receptors, Pituitary Hormone