Efficient application of a baculovirus-silkworm larvae expression system for obtaining porcine circovirus type 2 virus-like particles for a vaccine
Efficient application of a baculovirus-silkworm larvae expression system for obtaining porcine circovirus type 2 virus-like particles for a vaccine
复制标题
杆状病毒-蚕幼虫表达系统在获得疫苗用猪圆环病毒2型病毒样颗粒中的高效应用。
DOI：
10.1007/s00705-020-04754-9
复制
发表时间：
2020-08-05
影响因子：
2.7
通讯作者：
Sun, Jingchen
中科院分区：
文献类型：
--
作者：
He, Qianhua;Cao, Zhenming;Sun, Jingchen
猪圆环病毒2型（Porcine circovirus type 2，PCV2）是引起猪病的主要病原。它是最小的单链DNA病毒，其基因组包含四个主要的开放阅读框架（ORF）。ORF2编码的主要结构蛋白Cap能在体外自组装形成病毒样颗粒（virus-like particles，VLP），并含有主要抗原决定簇。在本研究中，我们开发了一种高效的方法来获得VLP，并优化了纯化条件。在这种方法中，我们用杆状病毒感染的家蚕幼虫表达了带有6x His标签的蛋白Cap，以及E. coliBL21（DE3）原核表达系统。家蚕幼虫和E. coliBL21（DE3）。从家蚕幼虫中纯化的Cap蛋白在体外自组装成VLPs，而从细菌中纯化的Cap蛋白不能自组装。透射电子显微镜证实了VLP的自组装。使用酶联免疫吸附试验（ELISA）证明使用杆状病毒系统产生的VLP的免疫原性。并对纯化工艺进行了优化。结果表明，利用杆状病毒感染家蚕幼虫的表达系统是获得PCV2 VLP的良好选择，具有开发低成本高效疫苗的潜力。
Porcine circovirus type 2 (PCV2) is a major pathogen associated with swine diseases. It is the smallest single-stranded DNA virus, and its genome contains four major open reading frames (ORFs). ORF2 encodes the major structural protein Cap, which can self-assemble into virus-like particles (VLPs)in vitroand contains the primary antigenic determinants. In this study, we developed a high-efficiency method for obtaining VLPs and optimized the purification conditions. In this method, we expressed the protein Cap with a 6x His tag using baculovirus-infected silkworm larvae as well as theE. coliBL21(DE3) prokaryotic expression system. The PCV2 Cap proteins produced by the silkworm larvae andE. coliBL21(DE3) were purified. Cap proteins purified from silkworm larvae self-assembled into VLPsin vitro, while the Cap proteins purified from bacteria were unable to self-assemble. Transmission electron microscopy confirmed the self-assembly of VLPs. The immunogenicity of the VLPs produced using the baculovirus system was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Furthermore, the purification process was optimized. The results demonstrated that the expression system using baculovirus-infected silkworm larvae is a good choice for obtaining VLPs of PCV2 and has potential for the development of a low-cost and efficient vaccine.