Study on the expression,purification and apoptosis-promoting effect of fusion protein Tat-Vp3 within Bac-to-Bac bac-ulovirus expression system
Tan Ting
Liu Yun
Luo Haihua
Li Cui
Jiang Yong
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.
Keywords：Cell-penetrating peptidesApoptinBac-to-Bac baculovirus expression systemPoliferation rateApoptosis
Publication Date：2014-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：6( 7-12 )