Immunogenicity Of Recombinant Rabies Virus G Protein And Mouse IgG Fc Fragment In Baculovirus Expression System

Rabies is an acute,lethal neurological disease whose pathogen is rabies virus(RABV),which is found in most terrestrial mammals.Once clinical symptoms appear after infection,the mortality rate is 100%,which is a major public health problem in most developing countries and regions.According to estimates by the World Health Organization(WHO),about 60,000 people worldwide die from rabies every year.RABV encodes five structural proteins,of which G protein is the only protein that determines virulence and induces the production of neutralizing antibodies.Therefore,the study of G protein expression can lay the foundation for the construction of efficient and safe vaccines.In this study,the G protein extracellular domain of rabies virus wild-type GD-SH-01 and the mouse IgG2 a Fcfragment(CH2,CH3,Hinge)were ligated with a linker to construct a recombinant IgG molecule SHG-Fc/wt.The rabies virus GD-SH-01 G protein extracellular domain(SHG)was amplified by gene cloning as a control.Based on the wildtype SHG-Fc/wt,IgG 310 and 435 histidine were mutated to alanine,and the ability to bind to FcRn was lost as a mutation control.The three fragments were ligated to the expression vector p Fast Bac Dual to construct a recombinant expression plasmid.The recombinant plasmid was transformed into DH10 Bac E.coli to obtain the recombinant shuttle vector Bacmid,and transfected into Sf9 cells to obtain recombinant baculovirus.The virus was amplified to the third generation(P3),and the cells were seeded at MOI=1 to obtain three recombinant proteins.Analysis by SDS-PAGE and LC-MS/MS liquid phase mass spectrometry showed that the target protein was consistent with the theoretical amino-acid size and sequence.Western-blot results showed that the three recombinant proteins could produce specific bands after incubation with mouse IgG-Fcpolyclonal antibody,rabies positive serum and mouse His-tag monoclonal antibody;indirect immunofluorescence test results showed small Fluorescent spots can be seen after incubation of murine IgG-Fcantibody and His-tag antibody;indirect ELISA results show that three recombinant proteins can be produced by using rabies immunoglobulin and mouse serum after intramuscular immunization as antibodies.reaction.Therefore,the above experiments showed that the three recombinant proteins expressed were all immunogenic.In summary,this study fused the rabies virus G protein and mouse IgG2 a Fcfragment in Baculovirus Expression System,and purified the protein,and verified that the expressed recombinant protein was immunogenic.This experiment will lay the foundation for the study of the next mucosal immune mimic FcRn-mediated IgG transport translocation pathway,and provide a new idea for the research and preparation of rabies oral vaccine.