• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

可溶性猪PD-1蛋白体外增强PCV2感染PBMCs免疫效应研究

In Vitro Immunity Enhancement of Soluble Porcine PD-1 against PCV2Infecting PBMCs in Pigs

  • 摘要:
    目的 通过研究猪圆环病毒II型(porcine circovirus type II, PCV2)感染过程中程序性死亡受体1(programmed cell death protein 1, PD-1)及其配体(programmed cell death 1 ligands, PD-Ls)信号通路作用,寻找调节PCV2免疫抑制新途径,减少PCV2感染造成的经济损失。
    方法 首先,利用实验室构建的重组表达宿主菌pET32a-PD1/Rosetta进行诱导表达、纯化,获得可溶性猪PD-1蛋白(soluble PD-1, sPD-1)的诱导表达条件;进而利用此条件制备大量具有活性的猪sPD-1蛋白,体外作用于PCV2感染的猪外周血单个核细胞(peripheral blood mononuclear cell, PBMCs),分别通过CCK-8和流式细胞术检测PBMCs的增殖情况,间接免疫荧光法和RT-qPCR检测猪sPD-1蛋白对PCV2病毒载量的影响,RT-qPCR和ELISA检测猪sPD-1蛋白对免疫相关细胞因子IL-2、IL-12、IL-21、IL-17A和IFN-γ等的转录水平和分泌水平的影响。
    结果 通过诱导表达、纯化获得了高纯度、高活性的重组猪sPD-1蛋白。与未使用猪sPD-1蛋白处理的PCV2感染PBMCs组相比,10 μg·mL−1猪sPD-1蛋白PCV2的病毒载量下降至1000 copies·μL−1以下;CCK-8检测结果显示猪sPD-1蛋白处理组细胞增殖指数明显提高(P<0.05);流式细胞术检测发现猪sPD-1蛋白组的平均荧光强度(68.6±10.14)%,相对于PCV2病毒组(28.7±3.18)%明显增强(P<0.05);反转录荧光定量PCR和ELISA检测,发现猪sPD-1蛋白处理组的细胞因子IL-2、IL-12和IFN-γ的转录水平和分泌水平明显升高(P<0.05)。
    结论 猪sPD-1蛋白可以通过阻断PD-1/PD-Ls通路降低PCV2的病毒载量,明显促进PBMCs的增殖,增强PBMCs细胞因子的转录水平和分泌水平免疫反应。因此,猪sPD-1蛋白在体外可以增强PCV2感染PBMCs的免疫反应,进一步为病毒性疾病的防控提供理论依据。

     

    Abstract:
    Objective Roles of the programmed cell death protein 1 (PD-1) and its ligands (PD-Ls) in suppressing the immune response and the soluble porcine PD-1 (sPD-1) protein in inhibiting the porcine circovirus type II (PCV2) infection on pigs were investigated in search for a new approach to control the disease in animals.
    Method The recombinant host, pET32a-PD1/Rosetta, constructed in the laboratory was used for the extensive induction and purification to determine the sPD-1 expression conditions. Under which, a large amount of sPD-1 was purified to react with the peripheral blood mononuclear cells (PBMCs) infected by PCV2. The effects of PD-1 on the cells were measured by the indirect immunofluorescence and RT-qPCR, on the PBMC proliferation by CCK-8 detection and flow cytometry, and on the transcriptions and secretions of IL-2, IL-12, IL-21, IL-17A, and IFN-γby RT-qPCR and ELISA.
    Results Highly purified recombinant sPD-1 showing high activity was successfully obtained. The viral load in the PBMCs induced with sPD-1 at 10 μg·mL−1 were significantly lower than that in those infected by PCV2 in the absence of sPD-1 to below 1 000 copies·μL−1. Meanwhile, the PBMC cell proliferation index shown by CCK-8 significantly increased (P<0.05). The flow cytometric measurement on fluorescence intensity of (68.6±10.14)% on the PBMCs was significantly higher with the presence of sPD-1 than (28.7±3.18)% on the counterparts without the soluble protein (P<0.05). The RT-qPCR and ELISA also showed the cytokine transcriptions and secretions of IL-2, IL-12, and IFN-γ to be significantly higher under the same condition (P<0.05).
    Conclusion The presentation of sPD-1 significantly inhibited PCV2 infection on PBMCs through the related metabolic pathways. At the same time, it promoted the PBMC cell proliferation, cytokine transcriptions, and secretion. Therefore, the protein could potentially be used in mitigating PCV2 infection on animals.

     

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