以新型杆状病毒载体在昆虫、哺乳动物细胞中表达克里米亚-刚果出血热病毒核蛋白基因

Use of a novel baculovirus vector to express nucleoprotein gene of Crimean-Congo hemorrhagic fever virus in both insect and mammalian cells

目的 构建可以在哺乳动物细胞中高效表达的新型杆状病毒载体 ,并利用其将克里米亚 刚果出血热病毒 (CCHFV)中国分离株 (新疆出血热病毒 ,XHFV)BA8816 6的核蛋白 (NP)基因在昆虫和哺乳动物细胞中进行表达。方法 将人巨细胞病毒 (CMV)立即早期 (IE)启动子连接至杆状病毒载体pFastBac1多角体启动子下游形成新载体pCB1,然后将XHFVNP基因克隆至该载体 ,通过重组质粒转染和病毒感染 ,检测其在哺乳动物细胞 (COS 7和Vero)及昆虫细胞中的表达。结果 连接至pCB1的XHFVNP基因均能在相应的细胞中获得良好表达 ;以重组杆状病毒感染的Vero细胞可以作为抗原检测XHF血清 ,与ELISA的检测结果完全一致 ,并与临床诊断有很好的平行性。结论 新型杆状病毒载体能够驱动外源基因在昆虫和哺乳动物细胞中高效表达 ,不仅能方便快速地制备诊断抗原 ,还具有发展重组病毒疫苗和基因治疗的潜力。

Objective To construct a novel baculovirus vector which is capable of promoting the high yield expression of foreign gene in mammalian cells and to express by this vector the nucleoprotein (NP) gene of Crimean Congo hemorrhagic fever virus (CCHFV) Chinese isolate (Xinjiang hemorrhagic fever virus, XHFV) BA88166 in insect and Vero cells. Methods Human cytomegalovirus (CMV) immediate early (IE) promoter was ligated to the baculovirus vector pFastBac1 downstream of the polyhedrin promoter to give rise to the novel vector pCB1. XHFV NP gene was cloned to this vector and was well expressed in COS 7 cells and Vero cells by means of recombinant plasmid transfection and baculovirus infection. Results The XHFV NP gene in vector pCB1 could be well expressed in mammalian cells. Vero cells infected with recombinant baculovirus harboring NP gene could be employed as antigens to detect XHF serum specimens whose results were in good correlation with those of ELISA and in parallel with clinical diagnoses.Conclusion This novel baculovirus vector is able to express the foreign gene efficiently in both insect and mammalian cells, which provides not only the convenient diagnostic antigens but also the potential for developing recombinant virus vaccines and gene therapies.