pcDNA3.1(+)-CYP19-GFP真核表达质粒构建

Construction of eukaryotic expression plasmids containing green fluorescent protein gene and CYP19 WT or its variants

目的:构建pcDNA3.1(+)-CYP19-GFP、pcDNA3.1(+)-CYP19W39R-GFP、pcDNA3.1(+)-CYP19R264C-GFP与pcDNA3.1(+)-CYP19W39R-R264C-GFP真核表达质粒,并观察pcDNA3.1(+)-CYP19-GFP质粒在MCF-7和Bcap-37细胞中的表达。方法:①采用RT-PCR技术扩增CYP19 cDNA,插入pcDNA3.1(+)载体中,构建pcDNA3.1(+)-CYP19表达质粒;酶切pcDNA3.1(+)-CYP19(304bp BamHⅠ)-GFP和pcDNA3.1(+)-CYP19质粒,构建pcDNA3.1(+)-CYP19-GFP表达质粒;②以pcDNA3.1(+)-CYP19-GFP质粒为模板,采用定点突变技术,构建pcDNA3.1(+)-CYP19W39R-GFP和pcDNA3.1(+)-CYP19R264C-GFP及pcDNA3.1(+)-CYP19W39R-R264C-GFP表达质粒;③pcDNA3.1(+)-CYP19-GFP质粒通过脂质体介导转染MCF-7和Bcap-37细胞,荧光显微镜观察其在细胞中的表达。结果:①酶切鉴定及测序验证pcDNA3.1(+)-CYP19-GFP构建成功;②测序验证pcDNA3.1(+)-CYP19W39R-GFP和pcDNA3.1(+)-CYP19R264C-GFP及pcDNA3.1(+)-CYP19W39R-R264C-GFP构建成功;③在经转染的MCF-7和Bcap-37细胞中观察到较强的绿色荧光。结论:pcDNA3.1(+)-CYP19-GFP、pcDNA3.1(+)-CYP19W39R-GFP和pcDNA3.1(+)-CYP19R264C-GFP及pcDNA3.1(+)-CYP19W39R-R264C-GFP真核表达质粒已成功构建,并证实pcDNA3.1(+)-CYP19-GFP质粒能在MCF-7和Bcap-37细胞中表达,为进一步研究CYP19基因单核苷酸多态性的确切功能奠定基础。

Objective: To construct eukaryotic expression plasmids containing green fluorescent protein gene and CYP19 wild-type or its vatiants(W39R,R264C,W39R-R264C) and to observe its expression in MCF-7 and Bcap-37 cells.Methods: The aromatase WT cDNA sequence was obtained by RT-PCR amplification and cloned into the eukaryotic expression vector pcDNA3.1(+).pcDNA3.1(+)-CYP19-GFP plasmid was then used as the template for site-directed mutation to create variant constructs(W39R,R264C,W39R-R264C).pcDNA3.1(+)-CYP19-GFP was transfected and expressed in MCF-7 and Bcap-37 cells.Results: The construction of pcDNA3.1(+)-CYP19-GFP plasmid was confirmed by enzyme digestion and DNA sequencing.pcDNA3.1(+)-CYP19W39R-GFP,pcDNA3.1(+)-CYP19R264C-GFP,pcDNA3.1(+)-CYP19W39R-R264C-GFP plasmids were confirmed by DNA sequencing.The MCF-7 and Bcap-37 cells transfected with the pcDNA3.1(+)-CYP19-GFP plasmid expressed reporter gene of GFP.Conclusion: The eukaryotic expression plasmids have been constructed and expressed in MCF-7 and Bcap-37 cells successfully,which lays the foundation for the research of biological activities of CYP19 variant allozymes.