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Title: Establishment of CHO cell line expressing human MCHR2 gene and research of its molecular characteristics
Authors: Yuan, Chengfu
Bu, Youquan
Sengupta, Joyeeta
Yang, Junxia
Huang, Xiuning
Cheng, Li
Zhang, Qin
Yi, Faping
Liu, Geli
Song, Fangzhou
Keywords: Eukaryotic expression vector;Gene expression;MCHR2;Molecular characteristics
Issue Date: Nov-2009
Publisher: CSIR
Abstract: 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 lipofectamineTM2000, 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 (Bmax) of CHO cell line was 309.97±1.14 fM·mg-1protein and the dissociation constant (Kd 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.
Page(s): 862-870
ISSN: 0975-1009 (Online); 0019-5189 (Print)
Appears in Collections:IJEB Vol.47(11) [November 2009]

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