The Assemble Of Porcine Circovirus Virus Type 2 Virus-like Particles And The Study Of Its Stability

PCV2 is smallest, non-enveloped, single-stranded circular DNA viruses, which is currently found in the mammals, belonging to the genus Circovirus in the family of Circoviridae. Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus associated diseases (PCVAD), such as postweaning multisystemic wasting syndrome (PMWS). PCV2 infection is widespread, cause economically important diseases and severe immunosuppression in apparently healthy swine. At the same time the PCV2 often mixed infection whit other pathogens (such as Classical swine fever virus and Porcine reproductive and respiratory syndrome virus) in clinical which caused serious impact on swine industry production worldwide. VLPs mimic the structure of authentic virus particles they are more preferable for vaccine candidate. The aim of the study was to research prokaryotic expression preparation of PCV2 VLPs, and the study of PCV2 VLPs stability at the long-term storage.In this study, first synthetic PCV2b/1B subtype (GenBank:KF700357) capsid protein gene was clone into pET100, and overexpressed in E. coli BL21 (DE3). Subsequently, the soluble Cap was rapidly purified in one step by Ni+ affinity chromatography. Test purification of proteins is the PCV2 Cap protein by SDS-PAGE protein-electrophoresis and western.PCV2 Cap can self-assembly form VLPs in vitro an appropriate, the VLPs was show by transmission electron microscopy, we found the size (-17nm) and morphology was close to the wild PCV2. Furthermore, based on TEM, western blots and IFA, E. coli derived VLPs has the ability of invasion of PK15 cells, and identify PCV2 positive serum. Explore the effects of buffers, NaCl concentration and temperatures on the PCV2 VLP stability. We validated the use of transmission electron microscopy (TEM) for measuring the hydrodynamic diameter (-20 nm) of VLP, which was close to the VLP diameter (-17nm) as measured by dynamic light scattering (DLS).Using TEM and ELISA techniques, we found that PCV2 VLPs remained the best stable in buffer A with 0.5M NaCl at 4?. These data altogether proved the stability of EV71 VLP and suggested that the VLP is amenable to bioprocessing and storage.The results in our study provides a simple and effective molecular experiment model for PCV2 assembly and invasion mechanism research, and for the clinical serological detection PCV2 neutralizing antibodies, PCV2 VLPs candidate vaccine prepared provides the feasibility.