CCHFV S基因的重组杆状病毒的构建及鉴定

Construction and identification of recombinant baculovirus with CCHFV S gene

目的利用杆状病毒表达系统(baculovirus expression vector systems,BEVS)构建含有克里米亚-刚果出血热病毒(Crimean-Congo hemorrhagic fever virus,CCHFV)S基因的重组杆状病毒,并在sf9昆虫细胞中表达出核衣壳蛋白(nucleoprotein,NP)。方法将合成的CCHFV S基因定向克隆入杆状病毒转移载体pFastBac^TM Dual,获得重组的转移载体pFastBac^TM Dual-CCHFV-S。将其转化到E.coli DH10Bac^TM感受态细胞中,筛选得到重组杆状病毒杆粒rBV-Bacmid-S。用Cellfectin®II试剂将rBV-Bacmid-S转染入sf9昆虫细胞获得重组杆状病毒rBV-CCHFV-S。通过IFA和Western blot检测CCHFV S基因的表达。结果构建了含有S基因的rBV-CCHFV-S,在sf9昆虫细胞中成功表达CCHFV NP。结论利用BEVS可以成功表达CCHFV NP,这为研究CCHFV NP的生物学特性,研制特异性的治疗药物和预防控制疫苗奠定基础。

Objective To construct recombinant baculovirus containing the S gene of Crimean-Congo hemorrhagic fever virus(CCHFV)is constructed using baculovirus expression vector systems(BEVS)and express nucleoprotein(NP)in sf9 insect cells.Methods The recombinant CCHFV S gene was directionally cloned into the baculovirus transfer vector pFastBac^TM Dual to obtain the recombinant transfer vector pFastBac^TM Dual-CCHFV-S,which was transformed into E.coli DH10Bac^TM competent cells and screened the recombinant bacmid rBV-Bacmid-S.Recombinant baculovirus rBV-CCHFV-S was obtained by transfecting rBV-Bacmid-S into sf9 insect cells with Cellfectin®II reagent.The expression of the CCHFV S gene was detected by IFA and Western blot.Results rBVCCHFV-S containing S gene was constructed and CCHFV NP was successfully expressed in sf9 insect cells.Conclusions By using BEVS,CCHFV NP can be successfully expressed,which lays a foundation for the study of biological characteristics of CCHFV NP and develops of specific therapeutic drugs and preventive vaccines.