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目的在杆状病毒表达系统转染的昆虫SF-9细胞中表达诺如病毒(Noroviruses,NoV)的衣壳蛋白VP1,并验证其与Caco-2细胞的结合活性。方法利用杆状病毒表达系统转染的昆虫SF-9细胞进行诺如病毒VP1蛋白表达,用蔗糖密度梯度超速离心方法纯化,采用Western blot鉴定表达产物,采用流式细胞术检测NoV病毒样颗粒(Virus-like particles,VLPs)与Caco-2细胞的结合活性。结果重组质粒转染细胞表达产物经蔗糖密度梯度超速离心后进行SDS-PAGE,在分子质量单位55~70ku之间有3条蛋白带,与预期重组蛋白NoV-VLPs大小相符;Western blot分析各蛋白组分均能被兔抗NoV多抗血清识别;流式细胞仪分析显示,加入重组蛋白NoV-VLPs后荧光信号较阴性对照组显著增强,表明表达的重组蛋白NoV-VLPs能够与Caco-2结合,且3个组分的结合能力不同(P<0.05),以组分5与Caco-2细胞结合的能力最强(P<0.05)。结论 NoV-VLPs表达成功,并证明NoV-VLPs具有与Caco-2细胞结合活性,为NoV疫苗的开发和防治药物的研制奠定了基础。
Objective To express capsid protein VP1 of noroviruses (NoV) in insect SF-9 cells transfected with baculovirus expression system and verify its binding activity with Caco-2 cells. Methods The VP1 protein of norovirus VP-1 was expressed in insect SF-9 cells transfected with baculovirus expression system. The recombinant protein was purified by sucrose density gradient ultracentrifugation. The expression product was identified by Western blot. The expression of NoV virus-like particles was detected by flow cytometry Virus-like particles, VLPs) to Caco-2 cells. Results The expressed product of the recombinant plasmid transfected cells was subjected to ultracentrifugation by sucrose density gradient and then subjected to SDS-PAGE. There were 3 protein bands between 55 and 70 ku of the molecular mass units, which were consistent with the size of the expected recombinant protein NoV-VLPs. Western blot analysis of each protein NoV polyclonal antiserum was detected by flow cytometry. Fluorescence analysis showed that the expression of NoV-VLPs was significantly increased compared with that of the negative control, indicating that the recombinant protein NoV-VLPs could bind to Caco-2 (P <0.05). The binding ability of component 5 to Caco-2 cells was the strongest (P <0.05). Conclusion NoV-VLPs were successfully expressed and proved that NoV-VLPs possessed binding activity with Caco-2 cells, which lays the foundation for the development of NoV vaccine and the development of drug for prevention and treatment.