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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 efficiently express Rabies virus (RABV) glycoprotein (G) protein in silkworm chrysalis using Bac-to-Bac baculovirus expression system. Methods The G gene fragment was amplified from the total RNA of rabies virus CVS-11 strain and inserted into the donor plasmid pFastBacⅠ-G to construct the recombinant donor plasmid pFastBacⅠ-G. The recombinant plasmid was transformed into E. coli DH10Bac to construct the recombinant baculovirus shuttle plasmid Bacmid-G , Which was transfected into BmN cells to obtain recombinant baculovirus containing G gene. The recombinant virus was infected silkworm chrysalis, collected hemolymph, Western blot analysis. Results The recombinant baculovirus shuttle plasmid Bacmid-G was proved by PCR identification correctly. Rabies virus glycoprotein was correctly expressed in BmN cells infected with recombinant baculovirus and in silkworm chrysalis. The recombinant protein expressed in silkworm chrysalis can be combined with His-tagged Monoclonal antibodies bind specifically to rabies positive sera. Conclusion The recombinant rabies virus glycoprotein baculovirus was successfully constructed and expressed in silkworm pupa. The expressed product has good antigenicity.