Prokaryotic Expression Of Capsid Protein Of Type A Seneca Virus And Its Assembly And Effect Evaluation Of Virus-Like Particles In Vitro

Seneca virus A?SVA?is a new pathogen that causes vesicular disease in pigs found in recent years.It belongs to he genus Senecavirus in family Picornaviridae?the species Senecavirus A?and is shown to be most closely related to cardioviruses.The affected pigs exhibited acute lameness and vesicular lesions without mortality,and the piglet mortality rate is up to 30%⁷0%.In 2007,Senecavirus A-associated SIVD was first reported in several American countries,cases of related infections have been reported in a number of different countries and regions;In the beginning of 2015,the first case of SVA infection in China was identified in Guangdong province,characterized by vesicular lesions in sows and acute death of neonatal piglets.Due to the constant variation of SVA virulent genotypes and phenotypes,there may be a potential risk of a large-scale epidemic of the disease,so it is urgent to establish and develop prevental and control strategies,espectionally,the vaccine products for the disease.Virus-like particles,as a safe and efficient new vaccine,are similar to the natural viral particle capsid in structure,free from nucleic acid and anti-virus risk,and endowed with good immunogenicity by densely arranged B/T cell epitopes.As a candidate vaccine,they have been widely studied in the field of human and animal vaccines.In this study,E.coli was used as the expression vector to construct the recombinant expression plasmid of SVA capsid protein,and the obtained target protein was self-assembled in vitro,finally forming complete virus-like particles,laying a foundation for the preparation of new SVA vaccine.1.Construction of pSMA-VP0,pSMK-VP1 and pSMC-VP3 recombinant expression vectors and their low-dose induction expression:The recombinant plasmids p-UC57SVAV0,p-UC57SVAV1,p-UC57SVAV3 were used as templates to amplify SVA target genes VP0,VP1,VP3 They were cloned into prokaryotic expression vectors pSMK,pSMA,pSMC containing poly-histidine?His?and small ubiquitin-like protein?SUMO?,respectively.The positive plasmids after sequencing were named pSMA-VP0,pSMK-VP1,pSMC-VP3,respectively,and then co-transformed into BL21?DE3?.Finally,positive expression bacteria were screened by SDS-PAGE using low-dose induction expression method.2.Recombinant protein His-VP0,His-VP1,His-VP3 expression and protein purification:optimization of separation conditions to improve the efficiency of its protein expression induced expression conditions were optimized,including the induction temperature,inducers,IPTG concentration and inducing OD_600nm00nm value of the former,induction time,using the best expression condition after a large number of express nickel column affinity chromatography purification method is used to obtain high purity of target protein,and Western blotting-in detecting its response to the original.By SDS-PAGE shows express bacterium OD_600nm00nm value was 1.1,IPTG was 0.4 mmol/L,induction temperature and time was 20?and 14h.The results of western blotting show that recombinant protein His-VP0,His-VP1 and His-VP3 have a good immunogenicity.3.SVA-VLPs assembly and effect evaluation:Using the SUMO protease to remove the His-SUMO tag of the N-terminus of the fusion protein,the natural conformation of VP0,VP1,VP3 can be assembled in a suitable environment in vitro.Based on the existing research,the effects of different concentrations of ammonium sulfate solution on the assembly efficiency of SAV VLPs were investigated in vitro.It was concluded that the appropriate concentration of ammonium sulfate buffer could significantly promote the assembly of SVA VLPs.Data from nanoparticle size analyzer?DLS?,sucrose density gradient centrifugation?SDGC?and transmission electron microscopy?TEM?showed that three protein monomers could be successfully assembled into SVA-VLPs in 400 mmol/L ammonium sulfate buffer,and the diameter's size is about 30 nm,it's outline is clear and round hollow particles under electron microscope.The Western-blotting results show that the particles have good reactogenicity.To sum up,this study took His label and the SUMO tag pSMK,pSMA and pSMC as the carrier to build the pSMA-VP0,pSMK-VP1,pSMC-VP3 recombinant plasmids,successfully expressed by prokaryotic expression system of soluble SVA three structural protein VP0,VP1,VP3,after optimized it's expression codition,the native conformation VP0,VP1,VP3 protein assembled into VLPs in vitro,this study will provide the SVA virus-like particles produced in the vaccine to lay a certain foundation.