Establishment of CHO cell line expressing human MCHR2 gene and research of its molecular<i style=""> </i>characteristics

Abstract

The whole length of MCHR2 gene cDNA fragment was amplified by PCR using human fetal brain cDNA library as template. The<b style=""> </b>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^TM2000, 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 f<i style="">M</i>·mg^-1protein and the dissociation constant (K_dvalue) was 0.170±0.0006 n<i style="">M</i>. MCH could stimulate Ca²⁺release, its 50% effective concentration (EC₅₀)was 2.32±0.01 n<i style="">M</i>.<b style=""> </b>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.<b style=""></b>