The Expression, Characterization And Immunogenicity Of P Particle Of The GⅡ,4Norovirus Hunter504D Strain

Noroviruses are the most common cause of viral gastroenteritis，with the numbersof reported outbreaks peaking characteristically between November and March in thenorthern hemisphere. An increase in the number of globally reported norovirusoutbreaks was seen the past decade, especially for outbreaks caused by successivegenogroup II genotype4(GII.4) variants. NoV can infect people of all ages in the world.The CDC has estimated that noroviruses are responsible for at least23million cases offoodborne illness each year in the US, and surveillance data from the FoodborneViruses in Europe Network indicates>85%of viral gastroenteritis outbreaks thatoccurred between1995and2000could be attributed these viruses.Illness is usually self-limiting and symptoms, comprising acute onset vomitingand watery diarrhea, subside within one to three days. NoV outbreaks, which mayaffect hundreds of people and are notoriously difficult to control, are primarilyassociated with places where people are in close contact, for example hospitals andlong-term care facilities. Since vaccines against HuNoV are still under study,preventive measures to avoid norovirus infection include hand washing, ingestion ofsafe water and food and disposal of contaminated material to avoid spread of infection.Human NoVs cannot be effectively cultivated in cell cultures or infect smallanimals and therefore a subunit vaccine is a choice for NoVs. NoV VLPs assemblespontaneously when the capsid protein is produced in insect cells through recombinantbaculoviruses or other eukaryotic expression system. However, VLP production wasunsuccessful in a prokaryotic expression system. Another type of NoV subviralparticles, the protruding (P) particles, has been developed that could be a secondgeneration vaccine against NoVs. The P particles are formed by the P domain andrevealed the same antigenic types as VLPs. They are highly immunogenic and verystable and most importantly, they can be efficiently produced in Escherichia coli.The GII.4NoV and its variants has been a global epidemic strains and existpersistent infection. In resent years, the GII.4NoV caused the ourbreaks has been thepredominant strain which cause the ourbreaks in our country. In this study, we choose the P domin of the Hunter strains which popular between2004and2006for research.Ihope to get highly expressed P protein in prokaryotic expression system.through thestudy of P protein, we could provide the theorctical basis for NoV subunit vaccinedevelopment.In this study，the P domain was amplified by PCR with the synthetic GII.4NoVstrain Hunter sequences of open reading frames2as a template and cloned into theexpression vector pET28a(+)，construction of expression vestor(pET28a-P Particle),the constrcts P protein expressed in E. coli strain BL21. The recombinant proteins wereseparated on SDS–PAGE gel, to test the quality of the recombinant proteins. Analysisof the expressed products showed that the P domain encodes a protein with an apparentmolecular weight of35KD. the P protein was isolated by Ni-NTA affinitychromatography、Gel filtration chromatography、electron microscopy analysis showedthat when the P domain is expressed in E. coli, it forms different P Particles. We thenstudied the immune responses to the P particle in Balb/c and New Zealandrabbit,ELISA to detect NoV antibody in serum using P protein protein as antigen, inorder to show that NoV antibody in serum has neutralizing antibody activity, wemeasure the ability of the immune antisera from mice to block P particles binding toHBGAs.