title: [The assemblage, purification and characterization of EV71 VLPs expressed in baculovirus].
abstract: To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1 and 3CD genes of enterovirus 71(EV71), the P1 and 3CD genes were cloned into the same baculovirus shuttle vector (Bacmid). Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-P1-3CD into the insect cell line of S f9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected S f9 cells. Electron microscopy showed that the structural protein capsid P1 was cut by virus-encoded protease 3CD and assembled into EV71 virus like particles (VLPs) about 27nm diameter. Different values of MOI and time points of expression were compared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used S f9 cells with serum-free medium in CellSTACK-10 Culture Chambers to produce EV71 VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1 (39kD), VP0 (34kD) and VP3 (26kD) as well as the intact structure, which can be greatly used for further study in protein structure and genetic engineering vaccine research.
authors: Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (IVDC, China CDC) Beijing 100052, China. clsunrise@126.com
authors: Lei
authors: L
authors: Cao
authors: Yao
authors: Y
authors: Yi
authors: Jing-Dong
authors: JD
authors: Song
authors: Miao-Miao
authors: MM
authors: Tian
authors: Rui-Guang
authors: RG
authors: Tian
authors: Qing-Ling
authors: QL
authors: Meng
authors: Feng
authors: F
authors: Qiu
authors: Zhi-Yuan
authors: ZY
authors: Jia
authors: Sheng-Li
authors: SL
authors: Bi
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D029821:Enterovirus A, Human / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology* / Q000648:ultrastructure; D015870:Gene Expression; D018411:Spodoptera; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology* / Q000648:ultrastructure; D019065:Virus Assembly
chemical_list: D014764:Viral Proteins