诺如病毒病毒样颗粒的表达及其与Caco-2细胞的结合活性研究

Expression of NoV virus-like particles and analysis of their binding activity with Caco-2cells

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VPl，并芎令证其与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VPl蛋白表达，用蔗糖密度梯瞍超速离心方法纯化，采用Westernblot鉴定表达产物，采用流式细胞术检测NoV病毒样颗粒（Virus-likeparticles，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—VI-Ps大小相符；Westernblot分析各蛋白组分均能被兔抗NoV多抗血清识别；流式细胞仪分析届示，加入重组蛋白NoV—VI—Ps后荧光信号较阴性对照组显著增强，表明表达的重组蛋自NoV—VI．Ps能够与Caco-2结合，且3个组分的结合能力不同（P〈i0·05），以组分5与Caco-2细胞结合的能力最强（P〈O．05）。结论NoVVI．Ps表达成功，并证明NoV-VI—Ps具有与Caco2细胞结合活性，为N。V疫苗的开发和防治药物的研制奠定了基础。

Objectives To express the capsid protein VP1 of noroviruses （NOV） in insect SF 9 cells with a baculovirus expression vector system and explore its binding activity with Caco 2 cells. Methods The NoV ORF2 gene was ex pressed with a baculovirus expression vector system. After collection of the cell supernatants of SF-9 cells, the recombi nant proteins were purified by sucrose ultra-centrifugation and identified by SDS PAGE and Western blotting. The bind- ing patterns of NoV virus like particles （VLPs） with Caco 2 ceils were then analyzed with flow cytometry. Results The NoV VLPs were purified by sucrose-ultra centrifugation followed by confirmation with SDS-PAGE and Western blot- ting. There were 3 protein bands between 55--70 ku according to SDS-PAGE, indicating molecules in expected sizes in accordance with the protein. All 3 protein bands were detected by rabbit anti-Nov serum in a Western blot array. The binding of NoV VLPs to Caco-2 cells was determined using flow cytometry. The results of flow cytometry showed that NoV-VI.Ps groups a significantly more intense fluorescent signal than the negative controls, indicating binding of the ex pressed recombinant NoV-VLPs to Caco-2 cells. Moreover, binding activity differed for 3 protein constituents （P〈 0.05）, and Constituent 5 had the strongest binding to Caco 2 cells （P〈0.05）. Conclusion NoV-VLPs were success fully expressed and a Caco-2 cell binding assay was created. These data will help to explore the means of host adaptation by NoV and will also lay a foundation for the development of immunological diagnostic reagents and vaccines for NoV.