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

作　　者：邵喜英[1] 陈占红[1] 曹江[2] 方永明[3] 王晓稼[1] 

机构地区：[1]浙江省肿瘤医院化疗中心,浙江杭州310022 [2]浙江大学医学院附属邵逸夫医院中心实验室,浙江杭州310016 [3]浙江大学医学院附属第二医院肿瘤研究所,浙江杭州310009

出　　处：《浙江大学学报（医学版）》2011年第2期189-194,共6页Journal of Zhejiang University(Medical Sciences)

基　　金：浙江省自然科学基金资助项目(Y206856);浙江省医药卫生优秀人才与科学研究基金资助项目(2005QN002);浙江省医药卫生科学研究基金资助项目(2007B021)

摘　　要：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.

关 键 词：细胞色素P450酶系统/遗传学 质粒 遗传载体 真核细胞 CYP19基因 真核表达质粒 GFP 

分 类 号：R346[医药卫生—基础医学]