【摘要】 目的 利用Bac-to-Bac杆状病毒表达系统表达小鼠肝炎病毒N基因。方法 根据GenBank发表的小鼠肝炎病毒A59株N基因序列(AY700211),设计一对特异性引物,利用RT-PCR方法扩增出N基因的ORF,将目的片段克隆入pFastBacHTa中,构建重组转移载体pFastBHa-N。将重组质粒转化DH10Bac感受态细菌,与Bacmid发生位点特异性转座作用,获得重组穿梭载体Bacmid-N。通过脂质体将其转染昆虫细胞Sf9。结果 获得了重组杆状病毒rBN，并通过Western blot和IFA分析表明证实N蛋白在昆虫细胞中获得正确表达,且表达产物具有抗原性。结论 利用Bac-toBac杆状病毒表达系统成功表达了小鼠肝炎病毒N蛋白，为进一步进行建立诊断学方法的研究奠定了基础。更多还原

【Abstract】 Objective To express the MHV nucleocapsid(N) gene with Bac-to-Bac baculovirus expression system. Method According to N gene sequence of MHV A59 strain in Genbank, a pair of specific primers was designed and synthesized. The ORF of N gene was amplified by RT-PCR, and then inserted into the transfer vehicle pFastbacHTa to obtain the recombinant transfer vectors pFastBHa-N,which was used to transform into E.coli DH10Bac, producing specific transposition with Bacmid to yield the recombinant shuttle vehicles, Bacmid-N. Then the Bacmid-N was further used to transfect Sf9insect cells by lipofectin to generate recombinant baculovirus expressing N recombination protein.Results Recombinant baculovirus, rBN, was obtained. IFA and Western blotting detection confirn1ed that the gene had been expressed in sf9 cells, and the expression product could specially react to the sera of MHV. Conclusion The MHV N protein is expressed successfully by Bac-to-Bac baeulovirus expression system, which may lay foundation for development of diagnostic technique.更多还原