Expression And Antigenicity Detection Of Self-assembled Rabies Virus G Protein-Ferritin Nanoparticles In Bombyx Mori

Rabies is one of the known high lethal infectious diseases in the world.Rabies is a zoonotic disease caused by rabies virus(Rabiesvirus,RV),which seriousiy damages the central nervous system.Once clinical symptoms are apparent,mortality is nearly 100%.At present,there is no effective treatment,only through vaccination to prevent and control rabies.Traditional vaccines include inactivated vaccine,live attenuated vaccine and so on,this kind of vaccine has been used for a long time,and the technology is relatively mature,but they also have some safety risks,such as strong toxicity,large adverse reactions and rejuvenation of the virus strain,and the production cost is expensive,so it is not suitable for large-scale vaccination.As a new type of biogenic nanomaterial,ferritin(Ferritin,Fe)can not only self-assemble into spherical nanostructures,display fused antigen proteins on its surface,promote antigen recognition and uptake by antigen-presenting cells,thus inducing stronger immune response,but also has the advantages of good biocompatibility,strong stability,low toxicity,modification and so on.It can be used as an ideal for the presentation platform of antigens,especially polymerized antigens,for the development of novel subunit vaccines.Rabies virus glycoprotein(Rabiesvirusglycoprotein,RVG)is considered as the major protective antigen of rabies virus,which can induce the body to produce virus neutralizing antibody and is the first choice for the preparation of subunit vaccine.In this study,the extracellular domain of rabies virus G protein and Helicobacter pylori ferritin gene were designed and optimized,and the G protein extracellular domain was connected to the N-terminal of ferritin by Linker(SGG).The ferritin nanoparticle antigen fused with rabies virus G protein was expressed in E.coli and Bombyx mori respectively,and the antigenicity of the expressed product was determined.The specific research contents are as follows:The consensus sequence of G protein of rabies virus was obtained by comparison on NCBI.Then the extracellular domain of G protein and the gene sequence of ferritin were optimized and synthesized,and the RVG-Fe fusion gene was obtained by fusion PCR.RVG and RVG-Fe genes were cloned into p ET28 a expression vector and transformed into Rosetta(DE3)for inducion expression.The recombinant protein was detected by Westernblot and mass spectrometry.The results showed that RVG protein and RVG-Fe protein were successfully expressed in E.coli.Since the recombinant protein exists mainly in the form of insoluble inclusion bodies,it needs to be renatured by dialysis to restore its advanced structure.After negative staining and immunogold labeling of the renatured RVG-Fe protein,it was observed that the fusion protein could be assembled into nanoparticles with the size of about 30 nm under transmission electron microscope,and the G protein was located on the surface of the spherical structure.The purified and renatured RVG-Fe protein was injected intraperitoneally at the dose of 20 μg/mouse,and The antibody titer in the serum of mice was about 1:25600 after 2 weeks of second immunization,indicating that the obtained RVG-Fe fusion protein could induce specific antibodies against rabies virus.The obtained RVG-Fe fusion gene was cloned into the baculovirus transfer vector p VL1393,and then co-transfected into Bm N cells by liposome-mediated baculovirus transfer vector and DNA of the inactivated rescued baculovirus to obtain the recombinant virus re Bm Bac-RVG-Fe;then the recombinant virus was screened and purified to infect the silkworm,and the expression products were collected.Indirect immunofluorescence assay showed that RVG-Fe protein was successfully expressed in Bm N cells;the results of viral genome PCR,RT-PCR and Western blot showed that the fusion gene was successfully replicated,transcribed and expressed in silkworm.The expression level of fusion protein in silkworm was determined by ELISA,and the results showed that the expression level of fusion protein was more than 648 μg/m L.After negative staining and immunogold treatment,the purified RVG-Fe protein could be observed that the fusion protein was self-assembled into nanoparticles of about 30 nm under transmission electron microscope,and the G protein was located on the surface of the spherical structure,indicating that the fusion protein with advanced structure was successfully expressed in the silkworm.Mice immunized with RVG-Fe fusion protein and inactivated rabies virus vaccine could produce high levels of specific antibodies.Using the RVG protein expressed in prokaryotic system as the detection antigen,the serum antibody titer of mice was detected by ELISA,and the serum antibody titer of mice injected with RVG-Fe antigen was about 1:12800,which was equivalent to that produced by inactivated vaccine,and the oral immunization of RVG-Fe fusion protein could also induce mice to produce specific antibodies against rabies virus,and the antibody titer was about 1:3200.In this study,based on the self-assembly characteristics of ferritin,it was used as a platform for presenting rabies virus G protein,and Bombyx mori was used as a bioreactor to express ferritin nanoparticles fused with rabies virus G protein in Bombyx mori baculovirus expression system.The nanoparticles antigen can induce mice to produce specific antibodies against rabies virus by both injection and oral administration.This lays an experimental foundation for the large-scale production of rabies vaccine,broadens the train of thought for the production of a new type of rabies vaccine,and provides a feasible scheme for the development of oral rabies vaccine.