Preparation Of Norovirus VLP Vaccine From Insect Cells And Silkworm Bioreactors

Norovirus,also known as Norwalk virus（NVs）,belongs to the family calicivirus and is a non-enveloped,single,and positive-stranded RNA virus.Norovirus is the main viral pathogen causing sporadic cases and outbreaks of epidemic acute gastroenteritis worldwide.There is a high morbidity rate in people with weak resistance,such as children,the elderly,and people with immune disorders who are prone to death or chronic disease after infection with Norovirus.GII.4 Norovirus has dominated in the past few decades.During the winter of2014-2015,the emergence of a novel variant of GII.17 became the dominant virus in China,leading to a significant increase in acute gastroenteritis outbreaks.Norovirus,the most important foodborne disease worldwide,is recognized as one of the most important pathogens.In China,specific commercialized Norovirus vaccines have not yet been marketed.It is urgently needed to develop effective norovirus vaccines for human health and safety.Norovirus can self-assemble into RNA-free virus-like particles（VLP）,which is similar to natural virus particles in structure and antigenicity.In addition,the immunogenicity and receptor binding properties of VLP make it an effective target for the development of Norovirus vaccines.The purpose of this study is to prepare a VLP vaccine against Norovirus epidemic strains by using the efficient biological reaction system of our group,providing an effective protection product for the prevention and control of Norovirus.In this study,the latest reported GII.17 type strain NV HN01/2015（KT992785）of Henan was selected in this study.After the expression of VP1 protein in the traditional baculovirus insect cell expression system,the production of the norovirus VLP vaccine was further explored in a silkworm bioreactor.The GII17.VP1 gene sequence was synthesized,and then cloned into two baculovirus transfer vectors,p YBDM-IM,and p UCDM-XIGP,and then the recombinant baculovirus with double copies was further packaged and infected with Sf9 cells.WB assay showed that Norovirus VP1 protein was successfully expressed in Sf9 cells.Subsequently,we purified the VP1 protein by cobalt ion affinity chromatography,and identified it by SDS-PAGE gel electrophoresis.We found an expected positive band of the VP1 protein that displayed as a single,clear,and bright band in WB,and this protein has good solubility.Further electron microscope observation showed that VLP with a size of about 32 nm,which proved that the expressed VP1 protein could self-assemble to form VLP.Then we recombined the transfer vector into Bombyx mori Bm Mulit Bac competent cells,tried to express the VP1 protein in the baculovirus expression system.We achieved a successful package of the Bombyx mori Bm BV-IM-ph-GII17.VP1 recombinant baculovirus for infection.After the silkworm and Bm N cells were detected by WB assay,it was found that the VP1 protein was successfully expressed in the silkworm cells.Further,we immunized mice with the norovirus VLP expressed in this study,for the test of immunogenicity and found that the recombinant immune protein could induce an immune response in mice and induce the production of norovirus-specific antibodies.This study is the first to use the baculovirus silkworm bioreactor expression system to successfully produce Norovirus VLPs,which provides a new method for the production of Norovirus vaccines.