FMDV 3C蛋白酶基因的克隆及在昆虫细胞中的表达

Cloning and expression of 3C protease gene from foot-and-mouth disease virus in insect cell

摘要:

口蹄疫病毒3C蛋白酶在病毒的致病机理、聚蛋白前体的加工和RNA的复制上起着很重要的作用,是当前抗病毒研究的一个重要靶点。本研究从AsiaⅠ型 FMDV适应细胞毒中提取RNA, 用RT-PCR技术扩增3C基因, 首先克隆到pGEM-T载体, 再亚克隆到杆状病毒转移载体pMelBac B中, 构建出重组转移载体pMel-3C。最后将含有目的基因的转移载体与线性化的杆状病毒DNA共转染Sf9细胞, 通过噬斑筛选和PCR鉴定, 获得了重组杆状病毒。重组病毒经扩增后以10个MOI感染Sf9细胞, 接种病毒72 h后收获细胞, 样品经SDS-PAGE和Western blot证实3C蛋白获得表达, 分子量约23kDa, 与预测蛋白大小一致, 且能被FMDV感染阳性血清所识别。本研究为空衣壳的体外组装及新型抗病毒药物设计的研究奠定了基础。

Abstract:

The 3C protease from foot-and-mouth disease virus (FMDV 3Cpro) is critical for viral pathogenesis, has vital roles in both processing of the polyprotein precursor and RNA replication, and is a potential anti-viral drug target. In the study, 3C gene of FMDV from serotype Asia I was obtained through Polymerase Chain Reaction (PCR), and subcloned into baculovirus transfer vector pMelBac-B. The recombinant transfer plasmid and linearized baculovirus DNA were co-transfected into sf9 insect cell, and the recombinant baculovirus were screened by plaque cloning and PCR identifica-tion. After amplification of recombinant baculovirus on cell passages, the recombinant virus were seeded on sf9 cell with 10 multiplicity of infection (MOI), and cells were harvested 72 hours after infection. The expressing product of 3C gene in insect cells was detected by Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and Western blot. The result demonstrated that the 3C gene was successfully expressed in insect cells. The product was a 23 kDa pro-tein and could be recognized by anti-FMDV serum in western blot. The results provide a basis for research of the assembly of FMDV empty capsids in vitro and the design of antivirus drug.