Expression And Identification Of Ferritin Nanoparticle Antigen Of Foot-and-mouth Disease Virus

Foot-and-mouth disease is an acute,hot,and contact animal infectious disease caused by foot-and-mouth disease virus and mainly directed against Artiodactyla.Seriously affected the development of animal husbandry and international trade in animal commodities.FMDV is a virus with strong infectivity,diverse transmission modes,sustainable infection and easy mutation,which brings great difficulty to prevention and elimination of the virus.In this study,ferritin nanoparticles can be modified to self-assemble.The silkworm baculovirus expression system is used as a new platform for vaccine development and antigen presentation.The foot-and-mouth disease virus O-type VP1 protein is presented to the outer surface of the ferritin shell.A strong immune response is induced,which lays the foundation for obtaining a broad-spectrum,safe and efficient FMD nanoparticle genetic engineering vaccine.This paper selects the VP1 amino acid sequence of the 10 most representative FMDV strains,and selects the VP1 amino acid sequence of KY399466.1 from the sequence analysis.The ferritin subunit is selected from the 5-167 amino acid sequence of Helicobacter pylori ferritin（NP₂23316）,and the ferritin gene is subjected to point mutation（N19Q）to eliminate the glycosylation site,and the SGG linker is added to the N end of the ferritin amino acid sequence.Used to connect the N-terminus of ferritin to the C-terminus of VP1.The silkworm codon optimization and synthesis were carried out on these two sequences.The vp1 gene sequence of the foot-and-mouth disease virus and the ferritin gene sequence were reconstructed into the vp1-ferritin gene sequence by fusion PCR.The fusion gene sequence and the foot-and-mouth disease virus vp1 gene sequence were cloned into the prokaryotic expression vector pET-28a（+）His tag sequence downstream to form pET-28a-VP1-Ferritin and pET-28a-Ferritin.The46.3 kDa recombinant protein His-VP1-Ferritin and the 27.4 kDa recombinant protein His-VP1 were successfully expressed in E.coli.After purification by affinity chromatography,Western blotting and protein mass spectrometry were performed,and the results were correct.The purified product His-VP1-Ferritin was used to prepare a vaccine to immunize mice.The antibody produced by enzyme-linked immunosorbent assay has a titer of 1:800,which proves that the His-VP1-Ferritin fusion gene has a good immunogen Sex.The two purified recombinant proteins were renatured and observed by transmission electron microscopy.It was found that the size of the particles assembled by His-VP1-Ferritin was about 20 nm,which was larger than the 12 nm of ferritin nanoparticles,while His-VP1 and blank control did not Appeared,it was preliminarily inferred that the VP1 structural protein was assembled to the outer surface of the ferritin cage structure,resulting in an increase in volume.Using the same method,the vp1-ferritin gene sequence and vp1 gene sequence were cloned into the downstream of the polyhedrin gene promoter of the transfer vector pVL-1393 of the baculovirus expression system.They were co-transfected with the ORF1629-deficient virus BmBacmid DNA intosilkworm cells,and homologously recombined into active recombinant baculovirus in the cells.Injecting silkworm larvae or silkworm pupae for expression,using the prokaryotic expression of the recombinant protein VP1-Ferritin to prepare mouse antibodies for Western blotting detection,there are obvious bands in the corresponding positions.After purification by ultracentrifugation,the negative staining method was used to observe through transmission electron microscopy.Similarly,a self-assembled particle structure of about 20 nm was found in the VP1-Ferritin sample,but not in VP1 and the blank control.The VP1-Ferritin and VP1 protein samples expressed by silkworm pupa were prepared as vaccines.SPF mice were immunized by injecting 10 mg of silkworm pupa fluid per mouse.After two immunizations,the mouse serum was collected.The titer of VP1-Ferritin antibody detected by enzyme-linked immunosorbent assay is not less than 1:1,600.Through experiments,it can be proved that immunization of mice expressing VP1-Ferritin recombinant protein through the silkworm baculovirus expression system can produce specific antibodies with very good immunogenicity.