摘要
目的:构建WFDC2启动子荧光素酶报告基因重组质粒PGL3-WFDC2-Promoter,并进行功能鉴定。方法:通过PCR的方法,从HO8910细胞基因组DNA中获得WFDC2基因编码序列上游的WFDC2启动子序列;软件分析WFDC2启动子的结构,选取调控密集区域,克隆到报告基因pGL3载体上,构建WFDC2启动子的报告基因载体;用脂质体转染法将2个报告基因载体分别转染至卵巢癌HO8910细胞系;采用双荧光素酶报告基因系统评估WFDC2启动子活性。结果:PCR方法成功钓取WFDC2启动子片段,测定其编码序列及读框正确,酶切鉴定亚克隆序列正确,瞬时转染HO8910后经报告基因检测,两段启动子均具有转录活性。结论:成功构建了WFDC2启动子的报告基因载体,为进一步研究WFDC2启动子和卵巢癌的关系奠定了实验基础。
Objective： To construct WFDC2 promoter luciferase reporter gene recombinant plasmid PGL3 - WFDC2 - Promoter, carry out functional identification. Methods： PCR was used to obtain WFDC2 promoter sequence at upstream of WFDC2 gene coding sequence from genome DNA of HO8910 cells; the structure of WFDC2 promoter was analyzed by a software, the intensive region of regulation was selected and cloned into PGL3 vector of reporter gene, then the reporter gene vector of WFDC2 promoter was constructed; liposome transfection was used to transfect two reporter gene vectors into ovarian cancer HO8910 ceils, respectively; the activity of WFDC2 promoter was evaluated by dual luciferase reporter gene system. Results： WFDC2 promoter fragments were obtained by PCR, the coding sequence and reading frame were correct, and the subclone sequence identified by enzyme digestion was correct, two promoters had transcriptional activity by reporter gene detection after transient transfeetion of HO8910 ＇cells. Conclusion： The WFDC2 promoter reporter gene vector is constructed successfully, which lay a experimental foundation for further research the relationship between WFDC2 promoter and ovarian cancer.
出处
《中国妇幼保健》
CAS
北大核心
2011年第25期3946-3948,共3页
Maternal and Child Health Care of China
基金
广东省卫生厅医学科研基金〔A2006375〕
