摘要
构建鼠OAZ1基因真核表达质粒,观察OAZ1-GFP融合蛋白在绿猴肾细胞COS7中的表达。利用RT-PCR的方法获得鼠OAZ1基因,再利用重叠延伸PCR缺失掉OAZ1序列中的205位碱基T,由此获得无需阅读框移码即可编码全长OAZ1的突变基因。将此突变基因克隆入真核表达载体pEGFP-N1获得重组质粒pEGFP-N1-OAZ1-T。将此质粒瞬时转染COS7后,采用RT-PCR,Western blotting,免疫荧光技术观察检测目的基因的表达情况。结果显示,酶切和测序证明质粒pEG-FP-N1-OAZ1-T构建正确,转染COS7细胞后,OAZ1-GFP融合蛋白能在细胞中高效表达。成功构建了pEGFP-N1-OAZ1-T真核表达质粒,在COS7细胞内,该质粒能成功指导OAZ1-GFP融合蛋白合成。
It was to construct eukaryotic expression plasmid of mouse OAZ1 and investigate its expression in COS7 cells.OAZ1 cDNA was amplified by RT-PCR from total RNA of mouse melanoma B16-F1 cells.The mutated OAZ1 gene that can translate whole-length OAZ1 without frame shifting was obtained by PCR-driven overlap extension and then subcloned into the plasmid pEGFP-N1.The recombinant plasmid pEGFP-N1-OAZ1-T which was identified by restriction enzyme digestion analysis and DNA sequencing,was transfected into COS7 cells by lipofectamine reagent transiently.The expression of OAZ1 gene in both mRNA and protein levels was confirmed by immunocytochemistry,RT-PCR and Western blotting analysis.Results showed that the OAZ1 gene was cloned and inserted into pEGFP-N1 successfully.In the COS7 cells,OAZ1-GFP fusion protein was expressed in high efficiency.The eukaryotic expression plasmid pEGFP-N1-OAZ1-T was successfully constructed and OAZ1-GFP fusion protein can be expressed in COS7 cells,which would lay a foundation for the further studies on the function of OAZ1.
出处
《生物技术通报》
CAS
CSCD
北大核心
2010年第9期173-176,180,共5页
Biotechnology Bulletin
基金
国家自然科学基金资助项目(30772590)
关键词
OAZ
多胺代谢
融合蛋白
真核表达
OAZ Polyamine metabolism Recombinant protein Eukaryotic expression
