pEGFP-N1-Mxi1-0重组质粒的构建与表达  

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

作　　者：胡圳圳[1] 李璐[2] 吴小芸[2] 郑大同[1,2] 吴伟玲[2] 

机构地区：[1]南京医科大学第二附属医院中心实验室,江苏南京210003 [2]南京医科大学第二附属医院儿童医学中心,江苏南京210003

出　　处：《蚌埠医学院学报》2015年第3期318-321,共4页Journal of Bengbu Medical College

基　　金：国家自然科学基金资助项目(81301822);南京医科大学科技发展基金重点资助项目(2012NJMU088);南京医科大学生殖医学国家重点实验室开放基金(SKLRMKF-1202)

摘　　要：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Hep G2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(p EGFP-N1)构建成p EGFP-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p EGFP-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p EGFP-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。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（ p EGFP-N1） to construct the recombinant vector p EGFP-N1-Mxi1-0. The recombinant vector p EGFP-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 p EGFP-N1-Mxi1-0 is successfully constructed. The Mxi1-0 expression in NIH /3T3 cells can be detected,which localizes in the cytoplasm.

关 键 词：基因表达 重组质粒 细胞定位 Max作用蛋白1-0 小鼠胚胎成纤维细胞 

分 类 号：Q343.1[生物学—遗传学]