利用昆虫杆状病毒载体表达人碱性成纤维细胞生长因子

目的利用Bac-to-Bac杆状病毒表达系统,将人碱性成纤维细胞生长因子(human basic fibroblast growth factor,hbFGF)基因进行表达,并检测重组蛋白的特异性及生物学活性。方法将人bFGF的cDNA克隆至杆状病毒转座载体pFastBacHTb中,并转化到含穿梭载体Bacmid的感受态细菌DH10Bac中,获得重组Bacmid/bFGF;纯化DNA后,转染昆虫细胞Sf21,PCR鉴定重组病毒;进一步将重组病毒感染Sf21细胞,在Sf21细胞中进行表达,对表达产物进行SDS-PAGE电泳、Western bloting分析;MTT法检测表达产物的生物活性。结果重组Bacmid/bFGFDNA直接转染昆虫细胞得到重组病毒rAcV-Bac-bFGF,病毒滴度MOI值≈8。重组病毒在感染后48h开始出现一相对分子质量为23000大小的特异带,感染后72h蛋白量明显增加,96h蛋白量达到最大,120h蛋白量开始减少。在正常昆虫Sf21细胞的对照中未见该蛋白带。Western bloting显示目的蛋白条带与人bFGF抗体发生特异反应。MTT结果显示重组蛋白能明显促进3T3细胞的分裂增殖。结论利用杆状病毒表达系统可成功表达具有生物学活性的bFGF蛋白。

Expression of Human Basic Fibroblast Growth Factor Gene in Baculovirus-Insect Expression System

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.