关键词: 人碱性成纤维细胞生长因子, Bac-to-Bac杆状病毒表达系统, 昆虫细胞, 基因表达

Abstract: Objective Bac-to-Bac baculovirus expression system was used to obtain biologically active recombinant human basic fibroblast growth factor(hbFGF) in insect cells. Methods The human bFGF cDNA was cloned into the transfer vector pFastBacHTb. The recombinant plasmid was introduced into E.coli DH10Bac which included a shuttle vector-Bacmid. The cultured Sf21 insect cells were directly transferred with the Bacmid/bFGF DNA, and the pure recombinant baculovirus rAcV-Bac-bFGF was obtained. Then rAcV-Bac-bFGF was infected Sf21 insect cells again to express the hbFGF gene, its expression product was measured by SDS-PAGE and Western blotting. The proliferation level of bFGF was detected by MTT. Results The target protein was detected at the time of 48 h post infection, reached to the peak at the time of 96 h post infection, and persisted expression until 120 h post infection. A 23 000 protein immunostaining band was detected in expressing protein with specific anti-hbFGF antibody by using Western blotting. MTT results showed that rhbFGF proteins had bioactivity to stimulate 3T3 cells proliferation. Conclusion Expression system of Bac-to-Bac baculovirus can be used successfully to express biological activity of hbFGF.

Key words: human basic fibroblast growth factor, Bac-to-Bac baculovirus expression system, insect cell, gene expression