肠道病毒71型收获液蛋白成分的分析

Analysis of protein component of harvested liquid of enterovirus 71

目的 分析肠道病毒71型（enterovirus71,EV71）收获液中的蛋白成分,为该类疫苗的研发及质控提供参考。方法 将EV71安徽阜阳株接种于Vero细胞,病毒培养72h后,收获病毒液,经蔗糖密度梯度超滤离心、甲醛灭活、DEAE Sephorase FF介质离子交换层析纯化,获得纯化样品。应用透射电镜分析样品中病毒颗粒的形态;对非还原型SDS-PAGE检测疑似病毒衣壳抗原蛋白VP1和VP3条带进行N-末端氨基酸序列分析;液相色谱（liquid chromatography,LC）与电喷雾电离质谱（electrospray ionization,ESI-MS）结合的方法分析样品中所含的蛋白成分。结果 纯化的EV71样品在透射电镜下可观察到20～30nm的球形EV71颗粒,呈典型的肠道病毒形态;经SDS-PAGE分析,第2和第3条带所在位置与文献报道的EV71衣壳蛋白VP1和VP3大小相符,N-末端氨基酸序列与GenBank报道的安徽阜阳株序列（ACD63039.1）一致;LC/ESI-MS质谱分析显示,纯化EV71样品中病毒蛋白成分占绝对优势,包括EV71的蛋白多聚体、VP1、VP2、VP3、VP4蛋白。结论 对EV71疫苗制备过程中病毒收获液的蛋白成分进行了初步分析,为该类疫苗的研发及质控提供了参考。

Objective To analyze the protein component in harvested liquid of enterovirus 71 （EV71） and provide a ref- erence for development and quality control of EV71 vaccine. Methods EV71 Anhui Fuyang strain was inoculated to Vero cells and cultured for 72 h, and the harvested liquid was concentrated by sucrose density gradient centrifugation combined with uhrafiltration, inactivated with formaldehyde and purified by DEAE Sepharosc FF ion exchange chromatog- raphy. The morphology of virus particles in test samples was analyzed by transmission electron microscopy, while the amino acids at N-terminus of suspected virus capsid protein VP1 and VP3 bands determined by non-reduced SDS-PAGE were sequenced, and the protein component by liquid chromatography （LC） and electrospray ionization （ESI-MS）. Re- suits Spheroidal particles at sizes of 20 ~ 30 nm were observed in purified EV71 samples under transmission electron microscope, which showed typical morphology of enterovirus. SDS-PAGE showed that the locations of the 2nd and 3rd bands were consistent with those of VPl and VP3 of EV71 reported respectively. The amino acid sequences at N-terminus of the two bands were consistent with that of EV71 Anhui Fuyang strain reported in GeuBank （ACD63039. 1 ）. LC/ESI- MS showed that viral protein components were prominent in purified EV71, including polymer, VP1, VP2, VP3 and VP4. Conclusion The protein component of virus liquid during preparation of EVT1 vaccine were preliminarily analyzed, which provided a reference for development and quality control of the vaccine.