Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi (KMUTT), Bangkok, 10140, Thailand.
Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Bangkok, 10400, Thailand; National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathum Thani, 12120, Thailand.
Protein Expr Purif. 2021 Aug;184:105876. doi: 10.1016/j.pep.2021.105876. Epub 2021 Mar 21.
Infectious spleen and kidney necrosis virus (ISKNV) is a causative agent of high mortality in fish resulting in significant economic loss to the fish industry in many countries. The major capsid protein (MCP) (ORF006) is an important structural component that mediates virus entry into the host cell, therefore it is a good candidate antigen of ISKNV for subunit vaccine development. In this study, MCP of ISKNV was successfully produced in Escherichia coli strain Ril and was purified as the soluble form by refolding recombinant MCP using urea in combination with dialysis process. The refolded recombinant MCP protein had ability of oligomerization to become trimer like native MCP protein. Fish immunized with refolded recombinant MCP showed significantly higher serum antibody titer than fish immunized with insoluble form of the protein (p < 0.05) at 21, 28- and 35-day post-immunization (dpi). Analysis of immune-related genes response in spleen and kidney of fish immunized with refolded recombinant MCP suggested that MHC-I, MHC-II, IL-1β and IL-4 genes were also significantly expressed relative to the group immunized with insoluble protein (p < 0.05) at 14, 21, 28- and 35-day post immunization. The highest serum antibody and immune related genes response were found at 28 day post immunization. Therefore, refolded recombinant MCP should be better than previously reported insoluble form as the candidate subunit vaccine to prevent infection of Nile tilapia from ISKNV.
传染性脾肾坏死病毒 (ISKNV) 是一种导致鱼类高死亡率的病原体，给许多国家的鱼类养殖业造成了重大经济损失。主要衣壳蛋白 (MCP)（ORF006）是一种重要的结构成分，介导病毒进入宿主细胞，因此它是 ISKNV 亚单位疫苗开发的良好候选抗原。在本研究中，成功在大肠杆菌菌株 Ril 中生产了 ISKNV 的 MCP，并通过使用尿素结合透析过程对重组 MCP 进行复性来将其纯化可溶性形式。复性的重组 MCP 蛋白具有三聚体样的寡聚化能力，成为天然 MCP 蛋白的三聚体。与免疫不可溶形式的蛋白相比，用复性重组 MCP 免疫的鱼类在 21、28-和 35 天攻毒后具有显著更高的血清抗体滴度（p<0.05）。对用复性重组 MCP 免疫的鱼类脾脏和肾脏中免疫相关基因反应的分析表明，与免疫不可溶蛋白的组相比，MHC-I、MHC-II、IL-1β 和 IL-4 基因在 14、21、28-和 35 天攻毒后也显著表达（p<0.05）。在攻毒后 28 天发现血清抗体和免疫相关基因反应最高。因此，与之前报道的不可溶形式相比，复性重组 MCP 应该是更好的候选亚单位疫苗，以预防尼罗罗非鱼感染 ISKNV。
