PRRSV重组非结构蛋白NSP1表达及免疫效果评价

Prokaryotic expression and immune effect evaluation of PRRSV nonstructural protein NSP1

摘要:

目的 表达并获取猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV) NADC 30毒株的重组非结构蛋白NSP1，评估其在小鼠体内的免疫效果，并探究PRRSV非结构蛋白作为疫苗抗原的潜在应用价值。方法 利用原核表达系统表达PRRSV NADC30毒株的NSP1蛋白，纯化后通过Western blotting验证NSP1的体外表达及其抗体反应性；将纯化后的重组NSP1蛋白免疫小鼠，测定其在小鼠体内诱导的细胞免疫和体液免疫水平；通过病毒中和试验评价重组蛋白NSP1诱导的中和抗体水平。结果 将NSP1目的基因连接至大肠杆菌pET-28a载体并进行原核表达，Western blotting结果显示，重组非结构蛋白NSP1能够正确表达，并且具有抗体反应性。经鉴定正确的重组蛋白NSP1免疫小鼠后，能够提高小鼠脾淋巴细胞中IFN-γ和TNF-α的表达水平，刺激机体产生细胞免疫应答。同时，重组蛋白NSP1还能够显著增强小鼠脾淋巴细胞的增殖能力。ELISA检测结果显示，免疫小鼠血清中的特异性抗体水平升高，进一步分析特异性抗体亚型(IgG2a和IgG1)发现，重组蛋白NSP1刺激机体产生的免疫类型偏向于Th2型体液免疫；病毒中和试验表明，重组蛋白NSP1具有良好的病毒中和能力，在免疫后28 d时中和效价为1:37，42 d时中和效价为1:31，均显著高于PBS对照组，与商品化疫苗组无差异。结论 PRRSV重组非结构蛋白NSP1免疫小鼠后能够刺激机体产生细胞免疫应答与体液免疫应答，并且具有良好的病毒中和能力，为新型PRRSV疫苗的开发提供了潜在的新思路。

Abstract:

Objective To express the recombinant nonstructural protein NSP1 of the porcine reproductive and respiratory syndrome virus (PRRSV) strain NADC 30, evaluate its immune effect in mice, and explore the potential value of the nonstructural proteins of PRRSV as vaccine antigens.Methods The prokaryotic expression system was used to express NSP1 of NADC30. After purification, the expression and antibody reactivity of NSP1 in vitro were identified by Western blotting. After mice were immunized with NSP1, the levels of cellular and humoral immunity induced by NSP1 were measured. The level of neutralizing antibody induced by NSP1 was evaluated by the virus neutralization assay.Results The target gene of NSP1 was connected to the Escherichia coli pET-28a vector for prokaryotic expression. Western blotting identified that the recombinant protein NSP1 was correctly expressed and had antibody reactivity. After mice were immunized with the confirmed recombinant protein NSP1, the levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the spleen lymphocytes of mice were elevated, and cellular immunity was stimulated. At the same time, the recombinant protein NSP1 significantly improved the proliferation of spleen lymphocytes in mice. ELISA results suggested that the level of specific antibodies in the serum rose after immunization. Further analysis of the specific antibody subtypes (IgG2a and IgG1) produced showed that the type of immunity stimulated by recombinant NSP1 was biased to Th2 humoral immunity. In addition, the virus neutralization assay showed that the recombinant protein NSP1 had a good virus neutralization ability, with the neutralization titer of 1:37 on day 28 and 1:31 on day 42, which were significantly higher than those of the PBS control group and had no difference from those of the commercial vaccine group.Conclusion The recombinant nonstructural protein NSP1 of PRRSV can stimulate cellular and humoral immunity in mice and has a good virus neutralization ability, which provides a new idea for the development of next-generation PRRSV vaccines.