重组杆状病毒载体在哺乳动物细胞中的表达

In vitro gene transfer into mammalian cells by recombinant baculovirus vector

目的 探讨重组杆状病毒作为哺乳动物基因转导载体的可行性及其转导特点。方法 应用Bac-to-Bac杆状病毒表达系统制备重组杆状病毒Bac-GFP,以不同MOI的病毒和不同终浓度丁酸钠感染各哺乳动物细胞系。通过荧光倒置相差显微镜和流式细胞仪观察检测细胞转导率和绿色荧光蛋白(GFP)表达强度。结果 发现HEK293细胞中,Bac-GFP转导率和GFP表达强度随着MOI和丁酸钠浓度的提高而显著增高(P<0.01),报告基因GFP的表达第2d最高,至少可持续9d。Bac-GFP可感染不同种属不同组织的哺乳动物细胞系,但转导率有差异。结论 重组杆状病毒可以用于体外基因转导。

Objective To study the feasibility and the characteristics of recombinant baculovirus as mammalian gene transfer vector. Methods After the generation of recombinant baculovirus Bac-GFP according to Bac-to-Bac baculovirus expression system, mammalian cell lines were infected by Bac-GFP with different multiplicities of infection (MOI) and different concentrations of sodium butyrate. The transduced cell rate and mean fluorescence strength (MFS) were detected by fluorescence microscopy and flow cytometry. Results In HEK293 cell line, the transduced cell rate and MFS significantly increased with increasing MOI and concentration of sodium butyrate ( P < 0.01). Expression of the reporter gene GFP reached the highest level on the second day postinfection. Mammalian cell lines from different genus and different tissues can be infected by Bac-GFP, but their transduced cell rate is extremely different. Conclusion Recombinant baculovirus can be a useful vector for in vitro gene transfer.