Preparation of divalent antigen-displaying enveloped virus-like particles using a single recombinant Bombyx mori nucleopolyhedrovirus bacmid in silkworms
Preparation of divalent antigen-displaying enveloped virus-like particles using a single recombinant Bombyx mori nucleopolyhedrovirus bacmid in silkworms
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DOI：
10.1016/j.jbiotec.2020.08.002
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发表时间：
2020-11-10
影响因子：
4.1
通讯作者：
Park，Enoch Y.
中科院分区：
文献类型：
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作者：
Kato，Tatsuya;Machida，Yuki;Park，Enoch Y.
在利用家蚕核型多角体病毒(BmNPV)构建的杆状病毒表达系统中，家蚕被用作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，这需要一个困难的DNA操作过程。在本研究中，我们构建了含有Rous肉瘤病毒(RSV)Gag蛋白、犬新孢子虫表面抗原1(NcSAG1)和SAG1相关序列2 ofn三个表达框的重组BmNPV。经Gibson组装和Bac-to-Bac系统，命名为BmNPV/SAG-SRS-Gag和BmNPV/SAG-Gag-SRS。BmNPV/SAG-SRS-Gag在家蚕中获得表达，并对其进行了鉴定。用蔗糖密度梯度离心法和亲和层析法分别用RSV Gag蛋白纯化NcSAG1和NcSRS2。透射电子显微镜观察表明，RSV Gag形成直径为20~30 nm的病毒样颗粒(RSV-LP)。免疫电子显微镜分析表明，NcSAG1和NcSRS2均显示在RSV-LPs的表面。这些结果表明，用单一多顺反子策略在家蚕幼虫血淋巴中产生了显示两种不同蛋白的RSV-LPs。该表达平台可有效地产生多抗原展示的VLP，并有助于传染病疫苗的开发。
Silkworms have been used as a host for the production of recombinant proteins in a baculovirus expression system usingBombyx morinucleopolyhedrovirus (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 ofNeospora caninum(NcSAG1) and SAG1-related sequence 2 ofN. 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.