猪瘟病毒E^(rns)和E2基因在昆虫细胞中的表达研究

Expression of E^(rns) and E2 Genes of Classical Swine Fever Virus in Insect Cells

利用昆虫细胞/杆状病毒系统表达猪瘟病毒(classical swine fever virus,CSFV)Erns和E2基因,用于CSFV新型疫苗以及建立相关的血清学诊断方法等研究.从猪瘟病毒石门株血液样品中提取总RNA,对Erns及E2基因进行扩增,将产物回收后分别连接于T载体,酶切及测序分析后,将其亚克隆至真核表达转移载体pFastBac HT经重组筛选后获得杆状病毒重组质粒,重组质粒转染昆虫细胞sf9后连续传3~4代,分别收获细胞上清液及沉淀,用于SDS-PAGE及Western-blotting试验,检测重组Erns,E2基因表达蛋白.用抗组氨酸(His)单克隆抗体在表达细胞中检测到Erns重组的His标签蛋白;应用抗E2蛋白单克隆抗体在表达细胞及培养上清液中检测到E2基因表达蛋白.结果表明在杆状病毒系统中成功表达了CSFV Erns和E2蛋白.

The Erns and E2 genes of classical swine fever virus（CSFV） were expressed in insect cells/Baculovirus expression system to establish CSFV serological diagnostic method and develop CSFV subunit vaccine.The Erns gene and E2 gene of CSFV shimen strain were amplified from total the RNA of the blood sample,and purified products were cloned into T-easy vector.After identification with enzyme digestion and sequence analysis,two genes were subcloned into the transfer vector pFastBacHT.Recombinant plasmids were obtained after selection and then transfected into sf9 cell,the recombinant baculoviruses were harvested after 3-4 passages,and the supernatants and cell deposit were detected by SDS-PAGE and Western-blotting.The obtained recombinant Erns protein reacted with the anti-His tag protein antibody and the E2 protein with the anti-E2 protein monoclone antibody by Western-blotting and immunohistochemical analysis.The results showed the Erns gene and E2 gene were successfully expressed in the Bac-to-Bac expression system.