猿猴空泡病毒40病毒样颗粒的制备及纯化
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Production and Purification of Simian Vacuolating Virus40 virus-like particles
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    摘要:

    猿猴空泡病毒40(Simian vacuolating virus 40,SV40)属于乳多空病毒科(Polyomaviridae)多瘤病毒属.本研究将SV40的主要衣壳蛋白VP1通过Bac-to-Bac杆状病毒表达系统在昆虫细胞中大量表达,并自我装配成形态结构及免疫原性均与天然病毒粒子相同或相似的SV40病毒样颗粒(SV40 virus-like particles,SV40 VLPs),经表达条件优化及分子筛纯化,成功制备出高纯度的VLPs.聚丙烯酰胺凝胶电泳(polyacrylamide gelelectrophoresis,SDS-PAGE)结果可见大小约为46KDa的VP1特异性条带.间接免疫荧光试验(IFA)证实VP1蛋白能够与异硫氰酸荧光素(fluorescein isothiocyanate, FITC)标记的羊抗鼠抗体发生反应,出现明显的特异性绿色荧光,具有良好的抗原性.纯化产物在透射电镜下可见直径约45nm的病毒样颗粒,显示出成功组装了SV40 VLPs,免疫印迹试验(Western-blot)证明VLPs能够与人抗SV40阳性血清发生反应,具有良好的抗原性.

    Abstract:

    Simian virus 40 (SV40) is a member of the Polyomaviridae family .Express the structural protein VP1 of SV40 in Bac-to-Bac baculovirus expression system. The expressed protein will be self-assembled to SV40 virus-like particles (SV40 VLPs). Then it will be extracted and purified by ultracentrifugation and molecular sieve. VP1 specified band could be observed at 46KDa in polyacrylamide gelelectrophoresis (SDS-PAGE). Indirect immunofluorescence assay (IFA)proved that VP1 VLPs combined with fluorescein isothiocyanate(FITC)-labeled goat anti-mouse antibody to present green fluorescence. This indicated VP1 protein had good antigenicity. The purified VLPs could be observed under electron microscope with 45nm diameter, which indicated SV40 VLPs was successfully assembled. The Western-blot showed SV40 VLPs could react with human-derived anti-SV40 plasma, which also indicated VLPs had good antigenicity.

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引用本文格式: 刘瑞熙,李晓雨,杨硕,李小姣,杨志荣,刘兰军,冯甦. 猿猴空泡病毒40病毒样颗粒的制备及纯化[J]. 四川大学学报: 自然科学版, 2016, 53: 437.

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  • 收稿日期:2014-11-29
  • 最后修改日期:2015-04-11
  • 录用日期:2015-04-17
  • 在线发布日期: 2016-05-30
  • 出版日期: