Preparation And Identification Of Seneca Virus-like Particles

Senecavirus A(SVA)belongs to the genus Picornaviridae Senecavirus and is a single-stranded positive-strand RNA virus.SVA can cause porcine idiopathic vesicular disease(PIVD),blistering symptoms in the mouth,hoof,and nose of sick pigs..In addition,some piglets infected with SVA will suffer from drowsiness,diarrhea and other symptoms,and the mortality rate of piglets under the age of 7 days can reach 30%.Since 2015,the SVA epidemic has appeared in many countries in North America,South America and Asia.It is distributed in South China,Central China and Northeast China,which poses a huge threat to the development of China’s pig industry.Vaccines are an important tool for controlling the spread of epidemics and protecting susceptible animals,but so far,there are still no commercial SVA vaccines.Therefore,it is of great significance to develop a new vaccine as soon as possible to prevent and control the SVA epidemic in China.Traditional inactivated and attenuated vaccines have some disadvantages,such as weak immune effect,poor safety,and inconvenient transportation.Virus-like Particles(VLPs)are formed by the aggregation of Virus structural protein,with abundant epitopes and no Virus genetic information,and can be expressed by escherichia coli expression system in large quantities in vitro.This study used escherichia coli expression system to express some structural proteins of SVA,self-assembled to form VLPs in vitro environment,and explored its immunogenicity,so as to provide basic data for the preparation of commercialized SVA vaccine in subsequent studies.The specific research contents are as follows:The virus was isolated from the tissue samples of the sick pigs,and the whole genome sequence was amplified by RT-PCR and the sequence was submitted to Gen Bank with the registration number of MG428683.The new strain was named CH-GDYD-2017.Homology analysis and phylogenetic tree analysis of SVA sequences in recent years showed that the strain had the highest homology with domestic CH-GDLZ-01-2017 and CH-01-2017 strains.It can be seen from the phylogenetic tree that the domestic SVA strain is in the same evolutionary branch as the American strain,and there is currently no completely independent evolution of the SVA strain.The obtained CH-GDYD-2017 genome was used as a template,and the structural proteins VP1,VP2 and VP3 of SVA were amplified by RT-PCR;the prokaryotic expression of these three genes was constructed using p ET-32a(+)plasmid.The plasmid was expressed and the p ET-32a(+)-VP1,p ET-32a(+)-VP2,p ET-32a(+)-VP3 expression plasmid was successfully constructed.Prokaryotic expression of VP1,VP2 and VP3 protein was performed,soluble expression of VP2 protein was performed,and inclusion body dissolution and renaturation of insoluble VP1 and VP3 protein were performed to restore their solubility.The protein was purified by column to obtain highly purified soluble protein,which was confirmed to be purified VP1,VP2 and VP3 protein by western-blot method.We will make VP1,VP2 and VP3 protein self-assembly in different environments,using transmission electron microscopy(TEM),in front of the assembled protein concentration is 0.5 mg/m L,0.05 M or 0.01 M of PBS buffer environment,VP1,VP2 and VP3 protein can self-assemble to form 20～30 nm in diameter virus-like particles to form,shape size conform to the expectations theory,explain VP1,VP2 and VP3 protein self-assembly can be assembled in a certain environment forms virus-like particles.Mice were immunized with VLPs and serum was collected.The serum antibody level of the mice was detected by indirect ELISA method,and it was found that the antibody level was at a high level after immunization with VLPs,indicating that the assembled VLPs could stimulate the mice to produce a certain humoral immune response.The results of real-time PCR and immunohistochemistry of mouse immunoprotection experiments showed that VLPs immunization can effectively reduce the viral load of mouse tissues.In summary,this study successfully isolated a new SVA strain CH-GDYD-2017(Gen Bank: MG428683),analyzed the prevalence of SVA strains in recent years,and provided basic data for the study of SVA epidemiology.The soluble expression of SVA structural proteins VP1,VP2 and VP3 was successfully achieved,and the self-assembly of SVA in VLPs in vitro was achieved by using the escherichia coli expression system,and its immunogenicity was detected,providing basic data for the subsequent study of SVA vaccine with VLPs as antigen.