Silkworms have been used as a host for the production of recombinant proteins in a baculovirus expression system using Bombyx mori nucleopolyhedrovirus (BmNPV). To coexpress several recombinant proteins, a silkworm must be coinfected with several recombinant BmNPVs, which requires a difficult DNA manipulation procedure. In this study, we constructed recombinant BmNPVs containing three expression cassettes, Rous sarcoma virus (RSV) Gag protein, surface antigen 1 of Neospora caninum (NcSAG1) and SAG1-related sequence 2 of N. caninum (NcSRS2), by Gibson assembly and the Bac-to-Bac system, designated BmNPV/SAG-SRS-Gag and BmNPV/SAG-Gag-SRS. BmNPV/SAG-SRS-Gag was expressed in silkworms and characterized. NcSAG1 and NcSRS2 were purified with RSV Gag proteins using sucrose density gradient centrifugation and affinity chromatography. RSV Gag formed virus-like particles (RSV-LPs) at a diameter of 20–30 nm based on transmission electron microscopy (TEM). Immuno-TEM analysis showed that both NcSAG1 and NcSRS2 were displayed on the surface of the RSV-LPs. These results indicate that RSV-LPs displaying two different kinds of proteins were produced in the hemolymph of silkworm larvae by the single polycistronic strategy. This expression platform is efficient for generating multiantigen-displaying VLPs and facilitates the development of vaccines against infectious diseases.

家蚕已被用作使用家蚕核多角体病毒(BmNPV)在杆状病毒表达系统中生产重组蛋白的宿主。为了共表达几种重组蛋白，家蚕必须与几种重组 BmNPV 共感染，这需要困难的 DNA 操作程序。在这项研究中，我们构建了含有三个表达盒，劳斯肉瘤病毒（RSV）Gag蛋白，表面抗原1重组BmNPVs犬新孢子虫（NcSAG1）和SAG1相关序列2犬新孢子虫(NcSRS2)，由 Gibson 组装和 Bac-to-Bac 系统，指定为 BmNPV/SAG-SRS-Gag 和 BmNPV/SAG-Gag-SRS。BmNPV/SAG-SRS-Gag 在家蚕中表达并表征。NcSAG1 和 NcSRS2 用 RSV Gag 蛋白使用蔗糖密度梯度离心和亲和层析进行纯化。基于透射电子显微镜 (TEM)，RSV Gag 形成直径为 20-30 nm 的病毒样颗粒 (RSV-LP)。免疫 TEM 分析显示 NcSAG1 和 NcSRS2 均显示在 RSV-LP 的表面。这些结果表明，通过单一多顺反子策略，在家蚕幼虫的血淋巴中产生了显示两种不同蛋白质的 RSV-LP。该表达平台可有效生成展示多抗原的 VLP，并有助于开发针对传染病的疫苗。