Construction of eukaryotic expression vector of human MCHR2 and establishment of stably transfected cell line

    YANG Junª²Xia, SHI Hua, WEI Liª²Li, YUAN Chengª²Fu, CHEN Ji, YI Faª²Ping, MA Yongª²Ping, SONG Fangª²Zhou

    Department of Biochemistry and Molecular Biology, Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology, Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education, Department of Pharmacology, Chongqing Medical Univerª²sity, Chongqing 400016, China

    ¡¾Abstract¡¿ AIM: To construct the eukaryotic expression vector of human melaninª²concentrating hormone receptor 2 (MCHR2) and stably transfect HEK293 cells with it. METHODS: The fullª²length MCHR2 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and was inserted into eukaryotic expression vector pcDNA3.1(+). After identification of restriction digestion and PCR, the recombinant plasmid was transfected into HEK293 cells by lipofectamine. After screening culture by G418, a stablyª²transfected cell line was established, and the transcription and expression of the MCHR2 gene were identified by RTª²PCR, Western blot and immunofluorescence assay. RESULTS: The eukaryotic expression vector pcDNA3.1ª²MCHR2 was successfully constructed and the MCHR2 gene was transfected stably into HEK293 cells. A stablyª²transfected cell line was established and the MCHR2 gene was expressed successfully. CONCLUSION: The establishment of the stablyª²transfected cell line and the expression of the target gene provide a solid experimental foundation for further studies on the function of the MCHR2 gene. ......