College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea.
Mol Biotechnol. 2012 Mar;50(3):211-20. doi: 10.1007/s12033-011-9431-5.
Although, classical swine fever virus (CSFV) envelope glycoprotein E2 subunit vaccine has been developed using the baculovirus expression system, the expression of viral antigens in baculovirus-infected insect cells is often ineffective. Therefore, an alternative strategy to the traditional baculovirus expression system is needed that is more productive and effective. Here, we report a novel strategy for the large-scale production of a CSFV E2 in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed native polyhedrin and approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg/ml of hemolymph and 0.53 mg/larva at 6-days post-infection. Six-week-old female BALB/c mice that were immunized with the E2ΔC protein purified from solubilized recombinant polyhedra elicited CSFV E2 antibodies, which indicated that the CSFV E2ΔC protein from recombinant polyhedra was immunogenic. The virus neutralization test showed that the serum from mice that were treated with E2ΔC protein from recombinant polyhedra contained significant levels of virus neutralization activity. These results demonstrate that this strategy can be used for the large-scale production of CSFV E2 antigen.
虽然已使用杆状病毒表达系统开发了经典猪瘟病毒（CSFV）包膜糖蛋白 E2 亚单位疫苗，但杆状病毒感染的昆虫细胞中病毒抗原的表达通常效率低下。因此，需要一种更具生产效率和效果的替代传统杆状病毒表达系统的策略。在这里，我们报告了一种在感染杆状病毒的家蚕幼虫中大规模生产 CSFV E2 的新策略。我们构建了一种重组家蚕核型多角体病毒（BmNPV），该病毒可表达与 CSFV E2 的 N 端 179 个氨基酸（E2ΔC）一起的重组多角体。BmNPV-E2ΔC 感染的家蚕幼虫表达了天然多角体蛋白和大约 44kDa 的融合蛋白，这两种蛋白都可以被抗多角体蛋白和抗 CSFV E2 抗体检测到。电子显微镜和共聚焦显微镜都证明了重组多角体包含融合蛋白和天然多角体，其形态正常，并且含有 CSFV E2ΔC。BmNPV-E2ΔC 感染的家蚕幼虫中产生的 CSFV E2ΔC 抗原达到 0.68mg/ml 的血淋巴和 0.53mg/幼虫，在感染后 6 天。用从重组多角体中溶解的融合蛋白纯化的 E2ΔC 蛋白免疫的 6 周龄雌性 BALB/c 小鼠产生了 CSFV E2 抗体，表明重组多角体中的 CSFV E2ΔC 蛋白具有免疫原性。病毒中和试验表明，用重组多角体中的 E2ΔC 蛋白处理的小鼠血清中含有显著水平的病毒中和活性。这些结果表明，该策略可用于大规模生产 CSFV E2 抗原。
