应用Bac-to-Bac杆状病毒表达系统在蚕蛹中表达狂犬病病毒糖蛋白

Expression of glycoprotein of rabies virus in silkworm chrysalis by using Bac-to-Bac baculovirus expression system

目的利用Bac-to-Bac杆状病毒表达系统在蚕蛹中高效表达狂犬病病毒(Rabies virus,RABV)糖(G)蛋白。方法从狂犬病病毒CVS-11株总RNA中扩增G基因片段,插入供体质粒pFastBacⅠ-G中,构建重组供体质粒pFastBacⅠ-G,转化大肠杆菌DH10Bac,构建重组杆状病毒穿梭质粒Bacmid-G,将其转染BmN细胞,获得含G基因的重组杆状病毒。将重组病毒感染蚕蛹,收集血淋巴,进行Western blot分析。结果重组杆状病毒穿梭质粒Bacmid-G经PCR鉴定证明构建正确;狂犬病病毒糖蛋白在重组杆状病毒感染的BmN细胞中和蚕蛹中获得正确表达,在蚕蛹中表达的重组蛋白可与His标记的单克隆抗体和狂犬病病毒阳性血清特异性结合。结论成功构建了重组狂犬病病毒糖蛋白杆状病毒,并在蚕蛹中获得了表达,表达产物具有良好的抗原性。

Objective To highly express the glycoprotein （G） of rabies virus （RABV） in silkworm chrysalis by using Bac-to- Bac baculovirus expression system. Methods The gene was amplified from total RNA of rabies virus CVS strain and inserted into plasmid pFastBac I vector. The constructed recombinant plasmid pFastBac I -G was transformed to E. coli DH10Bac, and the result- ed recombinant bacmid shuttle plasmid Bacmid-G was transfected to BmN cells. Silkworm chrysalis was infected with the obtained recombinant baculovirus, of which the hemolymph was collected and analyzed by Western blot. Results PCR and proved that re- combinant baculovirus shuttle plasmid Bacmid-G was constructed correctly. The G protein of rabies virus was expressed correctly in BmN cells infected with recombinant baculovirus, which showed specific binding to the monoclonal antibody with His-tag and rabies virus-positive sera. Conclusion Recombinant baculovirus for expression of G protein of rabies virus was constructed successfully and expressed in silkworm chrysalis, and the expressed product showed exceUent antigenicity.