Construction of high efficient eukaryotic expression vector carrying human β3-adrenoceptor gene and its significance

Aim: A high efficient eukaryotic expression vector carrying human β3-adrenoceptor (AR) genes was constructed to investigate the function of human β3-AR and interaction between β3-AR and PPAR-γ2 gene. Methods: The paranephric fat tissue from a patient with kidney stones was disrupted and homogenized, total RNA was finally extracted by Trizol kit. Human PPARγ2 (hPPARγ2) gene and human β3-AR (hβ3-AR) gene cDNA were amplified by using RT-PCR and then subcloned into pcDNA 3.1 empty vector. The pcDNA 3.1(+)/hβ3-AR(Mutant and Wild Type) was transfected into SH-SY5Y cell by electroporation, G418 (300 mg·L-1) was added into the media after 48 h transfection, and medium was changed every 3-5 days. The expression level of β3-AR protein was detected by RT-PCR and Western blot. Results: The sequencing results of amplified target genes showed that the sequence of human PPAR-γ2 gene and human β3-AR gene were the same as that of hPPARγ2 gene and hβ3-AR in Genebank. The mutants of pcDNA 3.1 (+)/hPPARγ2 (Pro12Ala) and pcDNA 3.1 (+)/hβ3-AR plasmid (Trp64Arg) were successfully constructed. Anti-G418 cell clones were formed after 2-4 weeks of G418 selection. RT-PCR results and Western blot analysis showed that anti-G418 SH-SY5Y cells transfected with pcDNA 3.1 (+)/hβ3-AR had the DNA fragment of hβ3-AR gene. Conclusions: The plasmids for encoding hPPARγ2 and hβ3-AR cDNAs were successfully cloned and the eukaryotic expression vectors [pcDNA 3.1 (+)/h PPARγ2 and pcDNA 3.1 (+)/hβ3-AR] of hPPARγ2 and hβ3-AR wild type and mutant genes were also successfully constructed. SH-SY5Y cell lines which stably expressed β3-AR genes were achieved successfully.

Duke Scholars

Author Huihua Li Pathology