猪δ冠状病毒与猪流行性腹泻病毒的受体结合区融合表达及免疫原性分析

Fusion expression and immunogenicity of receptor-binding domains of porcine deltacoronavirus and porcine epidemic diarrhea virus

为了研发出一种可同时预防猪德尔塔冠状病毒(porcine deltacoronavirus,PDCo V)和猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)两种病毒病的有效二联亚单位疫苗,本研究将PDCo V的受体结合域(receptor binding domain,RBD)与PEDV的RBD基因进行串联,构建至真核表达载体p CDNA3.1(+)中,利用哺乳动物细胞Expi CHO^(TM)表达系统表达纯化出PDCo V-RBD-PEDV-RBD(简写为pd RBD-pe RBD)融合蛋白,以3种不同剂量(10、20和30μg)免疫小鼠,利用ELISA和流式细胞术评估该融合蛋白诱导的体液免疫反应和细胞免疫反应,利用病毒微量中和试验测定免疫小鼠血清对PDCo V或PEDV的中和效价。结果表明,3个不同剂量组在加强免疫后均诱发了高水平的Ig G抗体,不同剂量组间抗体水平没有明显差异,说明10μg的免疫剂量即可达到较好免疫效果。流式细胞术检测结果显示,免疫组的CD3^(+)CD4^(+)T细胞比例显著升高,CD3^(+)CD8^(+)T细胞比例相对较低,这符合亚单位疫苗主导机体产生体液免疫反应的预期。同时,对血清中细胞因子白细胞介素-2(interleukin-2,IL-2)、白细胞介素-4(interleukin-4,IL-4)和干扰素-γ(interferon-γ,IFN-γ)的含量进行了检测,结果表明该融合蛋白诱导了良好的体液免疫效果,同时还产生了一定的细胞免疫应答。中和试验结果表明,10μg免疫剂量所诱导的抗体即可在体外有效中和PDCo V和PEDV两种病毒,效价分别为1:179.25和1:141.21。上述研究结果表明,pd RBD-pe RBD融合蛋白10μg免疫剂量即可诱导高水平的体液免疫反应,诱导的抗体可同时中和PDCo V和PEDV两种病毒,说明pd RBD-pe RBD融合蛋白有希望成为一种能同时预防PDCo V和PEDV的有效二联亚单位疫苗。

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^(TM) expression system and purified.Mice were immunized with the fusion protein at three different doses(10,20,and 30μg).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 10μg could achieve the fine immune effect.The results of flow cytometry showed that the immunization groups demonstrated increased proportion of CD3^(+)CD4^(+)T cells and decreased proportion of CD3^(+)CD8^(+)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)-γin 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 10μg fusion protein neutralized both PDCoV and PEDV in vitro,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 10μg,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.