小鼠Gtf2h2基因的克隆及其在真核细胞中的表达  

Mouse Gtf2h2 gene cloning and expression in eukaryotic cells

作　　者：张心悦[1] 杨光平[1] 时兰英[1] 苏友强[1] 

机构地区：[1]南京医科大学生殖医学国家重点实验室,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2015年第8期1049-1054,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家重大科学研究计划项目(2014CB943200;2013CB945500);国家自然基金面上项目(31471351)

摘　　要：目的:克隆小鼠Gtf2h2基因并使其在真核细胞HEK293中稳定表达。方法 :采用小鼠卵母细胞c DNA为模板,经PCR扩增Gtf2h2后,利用新型In-Fusion分子克隆体系将该目的片段克隆至真核表达载体p CMV6-AC-3DDK和p CMV6-ANm Kate中。所得阳性克隆的质粒DNA在经限制性酶切电泳和测序鉴定克隆成功后,转染到HEK293细胞中进行表达。表达效果利用抗DDK和m Kate标签的抗体通过免疫荧光以Western blot的方法进行检测。结果:质粒DNA测序和酶切鉴定证明Gtf2h2基因克隆成功;Western blot检测到GTF2H2-3DDK和GTF2H2-m Kate融合蛋白在转染后的HEK293细胞内的表达;免疫荧光显示融合蛋白在转染后的HEK293细胞核内定位并呈颗粒状分布。结论:成功克隆了小鼠Gtf2h2基因并实现了其在真核细胞HEK293中的稳定表达,为进一步研究其功能和作用机制奠定了基础。Objective:To clone mouse gene Gtf2h2 and have it stably expressed in HEK293 cells. Methods:Gtf2h2 was amplified by PCR using mouse oocyte c DNA as template,and was cloned into the eukaryotic cell-expression vector p CMV6-AC-3DDK and p CMV6-AN-m Kate using the advanced In-Fusion cloning system. After verification by restriction digestion followed by electrophoresis and sequencing,the correctly cloned plasmid DNA was transfected into HEK 293 cells. The expression of GTF2H2-3DDK and GTF2H2-m Kate fusion protein was detected by both immunofluorescence and Western blot using the antibodies of DDK and m Kate tag proteins. Results:The correct cloning of Gtf2h2 was confirmed by restriction digestion and sequencing,and the expression of GTF2H2-3DDK and GTF2H2-m Kate fusion protein was detected by Western blot. GTF2H2 fusion protein was detected in the nucleus in a punctate manner. Conclusion:Mouse Gtf2h2 gene was successfully cloned and expressed in HEK293 cells,which lays solid foundation for further studies of its functional roles and mechanisms.

关 键 词：转录因子 Gtf2h2 In-Fusion克隆 HEK293 

分 类 号：Q78[生物学—分子生物学]