小鼠Gtf2h2基因的克隆及其在真核细胞中的表达

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cDNA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pCMV6-AC-3DDK和pCMV6-AN-mKate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mKate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-mKate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?

Mouse Gtf2h2 gene cloning and expression in eukaryotic cells

Objective:To clone mouse gene Gtf2h2 and have it stably expressed in HEK293 cells. Methods:Gtf2h2 was amplified by PCR using mouse oocyte cDNA as template,and was cloned into the eukaryotic cell-expression vector pCMV6-AC-3DDK and pCMV6-AN-mKate 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-mKate fusion protein was detected by both immunofluorescence and Western blot using the antibodies of DDK and mKate tag proteins. Results:The correct cloning of Gtf2h2 was confirmed by restriction digestion and sequencing,and the expression of GTF2H2-3DDK and GTF2H2-mKate 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.