Construction of eukaryotic expressing vector of human MCHR1 and establishment of stable transfectant HEK293 cell line / 西安交通大学学报(医学版)

ABSTRACT

Objective To construct eukaryotic expressing vector of human melanin-concentrating hormone receptor1(MCHR1) and transfect HEK293 cells so as to establish stable HEK293 cell line.Methods The full-length MCHR1 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and was inserted into pcDNA3.1(+).After the identification of digestion and sequencing on the recombinant pcDNA3.1(+)/MCHR1,we transfected the recombinant into HEK293 cell by lipofectamineTM2000.After screening culture by G418,stable transfected HEK293 cell line was established,and the expression of MCHR1 was identified by RT-PCR and Western blotting.Results The eukaryotic expression vector pcDNA3.1(+)/MCHR1 was constructed,stable transfected HEK293 cell line was established,and MCHR1 protein was expressed successfully.Conclusion The construction of the eukaryotic expression vector pcDNA3.1(+)/MCHR1 and the establishment of stable transfected HEK293 cell line have provided solid experiment foundation for further studies on the function of MCHR1.