Gateway技术构建转基因重组质粒pRP.EX3d-EF1A-LRP16-His-IRES-eGFP.pdfVIP

258 解放军医学院学报 Acad J Chin PLA Med Sch Mar 2014，35（3） Gateway 技术构建转基因重组质粒pRP.EX3d-EF1A-LRP16-His-IRES- eGFP 柏苗苗，王春萌，伍志强，梅　倩，李小雷，李　祥，赵亚力，韩为东 解放军总医院 基础医学所分子生物室，北京　100853 摘要 ：目的 构建转基因重组质粒 pRP.EX3d-EF1A-LRP16-His-IRES-eGFP，为转基因鼠的建立奠定基础。 方法 利用重 叠 PCR 技术扩增 attB1-LRP16-His-attB2，经 BP 反应，将 LRP16 基因插入到载体 pDown 上，pUp-EF1A、pDown-LRP16- His、pTail-IRES-eGFP 和 PRP.Des3d 经 LR 反应，将 LRP16 转移到 pRP.EX3d 上，转化 Stbl3 细胞，用氨苄西林进行抗性筛选， 经 PCR 鉴定后将阳性克隆送测序。结果 测序及酶切结果验证获得正确的 pRP.EX3d-EF1A-LRP16-His-IRES-eGFP 重组质 粒。结论 成功构建 pRP.EX3d-EF1A-LRP16-His-IRES-eGFP 重组质粒，可用于下一步 LRP16 转基因小鼠的构建。 关键词 ：LRP16 ；转基因小鼠 ；质粒 ；Gateway 技术 DOI ： 中图分类号 ：R 34　文献标志码 ：A　文章编号 ：2095-5227(2014)03-0258-03　 10.3969/j.issn.2095-5227.2014.03.018 网络出版时间：2014-01-10 14:54　　　 网络出版地址 ：/kcms/detail/11.3275.R1454.003.html Application of gateway technology in construction of recombinant transgenic vector pRP. EX3d-EF1A-LRP16-His-IRES-eGFP BAI Miao-miao, WANG Chun-meng, WU Zhi-qiang, MEI Qian, LI Xiao-lei, LI Xiang, ZHAO Ya-li, HAN Wei-dong Department of Molecular Biology, Institute of Basic Medicine, Chinese PLA General Hospital, Beij ing 100853, China Corresponding author: HAN Wei-dong. Email: hanwdrsw69 @ Abstract: Objective To lay the foundation for establishing transgenic mice by constructing recombinant transgenic vector pRP. EX3d-EF 1A-LRP 16-His-IRES-eGFP. Methods The attB 1- LRP 16-His-attB2 was amplified by overlap PCR. The LRP 16 gene was cloned into pDown vector via BP reaction and transmitted into pRP.EX3d vector via LR reaction with pDown-LRP 16-His, pUp-EF 1A, pTail- IRES-eGFP and PRP. Des3d. The recombinant plasmid was transformed into Stbl3 cells and screened using the ampicillin (AMP) resistance gene. Positive clones were confirmed by PCR and DNA sequencing, respectively. Results Sequencing and restriction endonuclease showed that the recombinant vector pRP.EX3