感染、炎症、修复Issue(1)：7-12,6.DOI:10.3969/j.issn.1672-8521.2014.01.002
Tat-Vp3融合蛋白在杆状病毒-昆虫细胞表达系统中的表达与纯化及其促凋亡功能的研究
Study on the expression,purification and apoptosis-promoting effect of fusion protein Tat-Vp3 within Bac-to-Bac bac-ulovirus expression system
摘要
Abstract
Objective:A Bac-to-Bac baculovirus expression system was designed to obtain His-Tat-Vp3 soluble fusion pro-tein,and verify its pro-apoptotic function and activity. Methods:The plasmid pFastBac1-TAT-VP3 was constructed and transformed into DH10 BacTM E. coli for the construction of baculovirus plasmid. Then Sf9 insect cells were transfected with the plasmid to obtain recombinant baculovirus,which was purified and amplified. Sf9 insect cells were stimulated with the vi-rus with the optimum titer in order to induce the expression of the fusion protein. The output of target protein with molecu-lar mass of about 21 kDa was identified by SDS-PAGE and Western blot. Finally,MTT assay and flow cytometry apoptosis assay were used to detect the inhibition of tumor cell (HeLa)proliferation and pro-apoptotic activity with 500,1 000,2 000 nmol/L fusion protein. Results:The Tat-Vp3 fusion protein was successfully expressed and purified by Bac-to-Bac Baculovir-us Expression System,in which 1 000,2 000 nmol/L of fusion protein group was found to be able to provide a high inhibi-tive effect on the proliferation of tumor cells,as well as to enhance pro-apoptotic activity. Conclusions:The fusion protein ex-pression vector His-TAT-VP3 is successfully constructed based on the Bac-to-Bac Baculovirus Expression System,through which a soluble recombinant protein is induced. The obtained fusion protein can significantly inhibit the proliferation of Hela cells and induce apoptosis. This finding lays an important foundation for further research targeting pro-apoptotic agents.关键词
细胞穿透肽/民凋亡素/杆状病毒-昆虫细胞表达系统/增殖率/凋亡Key words
Cell-penetrating peptides/Apoptin/Bac-to-Bac baculovirus expression system/Poliferation rate/Apoptosis引用本文复制引用
谭婷,刘芸,罗海华,李翠,姜勇..Tat-Vp3融合蛋白在杆状病毒-昆虫细胞表达系统中的表达与纯化及其促凋亡功能的研究[J].感染、炎症、修复,2014,(1):7-12,6.基金项目
国家自然科学基金委员会重点项目(81030055) （81030055）
广东省自然基金项目(S2013010014422,S2011010003917,10251051501000003) （S2013010014422,S2011010003917,10251051501000003）
教育部博士点基金项目(20104433110008) （20104433110008）
广东省医学科学技术研究基金面上项目(A2013354) （A2013354）
2012年南方医科大学科研启动计划(B1012008) （B1012008）
