摘要
目的建立能够稳定分泌表达TAT融合蛋白的哺乳动物细胞系。方法依次将合成的编码TAT蛋白转导域和EG-FP的DNA片段插入分泌型真核表达载体pSecTag2A,构建编码TAT-EGFP融合蛋白的重组质粒,转染Vero细胞后,经Zeocin筛选建系,通过荧光显微镜和免疫印迹鉴定表达的蛋白,MTT分析细胞毒性,荧光观察蛋白转导活性。结果成功获得了稳定分泌表达TAT-EGFP融合蛋白的工程细胞系,表达的TAT融合蛋白具有良好的蛋白转导活性。结论建立的哺乳动物细胞表达系统能够持续产生具有蛋白转导活性的TAT融合蛋白,为相关应用研究奠定了基础。
Objective To construct an eukaryotic expression vector system and develop mammalian cell lines stably expressing TAT- EGFP fusion proteins, Methods Two oligonucleotides were synthesized and annealed to generate a double-stranded oligonucleotide encoding the TAT protein transduction domain which was then inserted into the plasmid pSecTag 2A to obtain pSec-TAT. Then the EGFP gene fragments were amplified and inserted into pSec-TAT to obtain the desired expression plasmid pTAT -EGFP. A mammalian cell line Vero was transfected with the plasmid pTAT -EGFP by lipofection. Vero cells transfected with pTAT -EGFP were selected under the pressure of Zeocin, and resistant cell clones were then expansively cultured and identified by Western blotting and fluorescent microscopy. The secreted TAT fusion protein was further added to the media of cultured Vero cells to observe their ability to enter cells using fluorescent microscopy, and MTT assay was also performed to examine the cytotoxicity of the fusion protein. Results An eukaryotic expression vector encoding TAT-EGFP fusion protein was constructed. A Zeocin resistant cell clone was selected and expansively cultured, and the results of Western blotting and fluorescent microscopy demonstrated that an engineered cell line stably expressing the TAT -EGFP fusion protein was established, which was named Vero-TAT-EGFP. There was no discernible morphological difference between Vero-TAT-EGFP and Vero cells, and MTT assay suggested that the TAT-fusion protein had no cytotoxicity to mammalian cells. Also, the secreted TAT-EGFP fusion protein was found active to enter cells rapidly. Condusions This engineered cell line provides a useful tool for future research based on protein transduction domains and may help to devise novel strategies in the development of protein therapy.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2007年第10期1031-1033,共3页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金资助项目(30570084
30600530)
