Tong Lixin, Lu Yanzhen, Yu Ruiming, Zhang Liping, Liu Xia, Wang Yonglu, DU Xiaohua, Liu Xinsheng
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, Gansu, China.
State Key Laboratory for Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, Gansu, China.
Sheng Wu Gong Cheng Xue Bao. 2024 Jul 25;40(7):2150-2161. doi: 10.13345/j.cjb.230826.
This study aims to develop an effective bivalent subunit vaccine that is promising to prevent both porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV). The receptor-binding domains (RBDs) of PDCoV and PEDV were fused and cloned into the eukaryotic expression vector pCDNA3.1(+). The fusion protein PDCoV-RBD-PEDV-RBD (pdRBD-peRBD) was expressed by the ExpiCHO expression system and purified. Mice were immunized with the fusion protein at three different doses (10, 20, and 30 μg). The humoral immune response and cellular immune response induced by the fusion protein were evaluated by ELISA and flow cytometry. The neutralization titers of the serum of immunized mice against PDCoV and PEDV were determined by the microneutralization test. The results showed that high levels of IgG antibodies were induced in the three different dose groups after booster immunization, and there was no significant difference in the antibody level between different dose groups, indicating that the immunization dose of 10 μg could achieve the fine immune effect. The results of flow cytometry showed that the immunization groups demonstrated increased proportion of CDCD T cells and decreased proportion of CDCD T cells, which was consistent with the expectation about the humoral immune response induced by the subunit vaccine. At the same time, the levels of interleukin (IL)-2, IL-4, and interferon (IFN)-γ in the serum were determined. The results showed that the fusion protein induced both humoral immune effect and cellular immune response. The results of the neutralization test showed that the antibody induced by 10 μg fusion protein neutralized both PDCoV and PEDV , with the titers of 1:179.25 and 1:141.21, respectively. The above results suggested that the pdRBD-peRBD could induce a high level of humoral immune response at a dose of 10 μg, and the induced antibody could neutralize both PDCoV and PEDV. Therefore, the fusion protein pdRBD-peRBD is expected to be an effective subunit vaccine that can simultaneously prevent PDCoV and PEDV.
本研究旨在开发一种有望预防猪德尔塔冠状病毒（PDCoV）和猪流行性腹泻病毒（PEDV）的有效二价亚单位疫苗。将PDCoV和PEDV的受体结合域（RBD）融合并克隆到真核表达载体pCDNA3.1(+)中。融合蛋白PDCoV-RBD-PEDV-RBD（pdRBD-peRBD）通过ExpiCHO表达系统表达并纯化。用三种不同剂量（10、20和30μg）的融合蛋白免疫小鼠。通过ELISA和流式细胞术评估融合蛋白诱导的体液免疫反应和细胞免疫反应。通过微量中和试验测定免疫小鼠血清针对PDCoV和PEDV的中和滴度。结果显示，加强免疫后三个不同剂量组均诱导出高水平的IgG抗体，不同剂量组之间抗体水平无显著差异，表明10μg的免疫剂量可达到良好的免疫效果。流式细胞术结果显示，免疫组CD4+T细胞比例增加，CD8+T细胞比例降低，这与亚单位疫苗诱导的体液免疫反应预期一致。同时，测定血清中白细胞介素（IL）-2、IL-4和干扰素（IFN）-γ的水平。结果显示，融合蛋白诱导了体液免疫效应和细胞免疫反应。中和试验结果显示，10μg融合蛋白诱导的抗体可同时中和PDCoV和PEDV，滴度分别为1:179.25和1:141.21。上述结果表明，pdRBD-peRBD在10μg剂量下可诱导高水平的体液免疫反应，且诱导的抗体可同时中和PDCoV和PEDV。因此，融合蛋白pdRBD-peRBD有望成为一种可同时预防PDCoV和PEDV的有效亚单位疫苗。
