pEGFP-N1-Mxi1-0重组质粒的构建与表达

目的:构建Max作用蛋白1-0(Max interacting protein1-0,Mxi1-0) 的真核表达载体,在小鼠胚胎成纤维细胞(NIH/3T3)中表达,以期为深入研究Mxi1-0的作用及机制奠定基础。方法:通过RT-PCR方法从人肿瘤细胞HepG2中获得Mxi1-0基因的编码片段,连接至增强绿色荧光蛋白真核表达载体(pEGFP-N1)构建成pEGFP-N1-Mxi1-0。经酶切和测序鉴定重组质粒的正确性;采用脂质体转染技术将重组质粒瞬时转染NIH/3T3细胞,经荧光显微镜观察和Western blot方法检测Mxi1-0表达,免疫荧光法检测Mxi1-0在NIH/3T3细胞内的定位情况。结果:经双酶切和核酸序列测序鉴定证实含Mxi1-0的重组真核表达载体pEGFP-Mxi1-0构建成功。重组质粒瞬时转染NIH/3T3细胞后,检测到Mxi1-0的成功表达,并证实Mxi1-0主要定位于NIH/3T3细胞质中。结论:成功构建了真核表达载体pEGFP-N1-Mxi1-0,并检测到Mxi1-0的表达,实验证明Mxi1-0定位于NIH/3T3细胞质中。

Construction and expression of the recombinant plasmid pEGFP-N1-Mxi1-0

Objective:To construct the recombinant eukaryote expression vector containing Max interacting protein(Mxi)1-0 gene,and detect the expression of Mxi1-0 in mouse embryo fibroblast(NIH/ 3T3) cells for providing the basis to explore the effect and mechanism of Mxi1-0. Methods:Mxi1-0 gene was cloned by RT-PCR from cancer cells,which was subcloned into enhanced green fluorescent protein eukaryote expression vector ( pEGFP-N1) to construct the recombinant vector pEGFP-N1-Mxi1-0. The recombinant vector pEGFP-N1-Mxi1-0 was identified by enzyme digestion and sequencing,which was transfected into the NIH/ 3T3 cells by lipidosome. The protein expression of Mxi1-0 in NIH/ 3T3 cells was detected by Western blot,the intracellular localization of Mxi1-0 was investigated by immunofluorescence. Results: The recombinant eukaryote expression vector encoding Mxi1-0 was successfully constructed. The expression of Mxi1-0 in NIH/ 3T3 cells could be detected by Western blot. The Mxi1-0 localized in the cytoplasm of NIH/ 3T3 cells. Conclusions:The recombinant expression vector pEGFP-N1-Mxi1-0 is successfully constructed. The Mxi1-0 expression in NIH/ 3T3 cells can be detected,which localizes in the cytoplasm.