Study On Rabies Virus Glycoprotein DNA Vaccine

Rabies is a global infectious disease caused by rabies virus,and exists in more than 150 countries and regions.According to the World Health Organization WHO 2016 statistics,about 59000 people died of the disease worldwide every year,of which 95%occurred in developing countries such as Asia and Africa.Rabies is an almost 100%fatal infectious disease.Therefore,it is necessary to develop rabies vaccine to prevent and control the spread of rabies virus.Viral glycoprotein(G)is the only rabies virus that can induce neutralizing antibodies.It can also stimulate the proliferation of T lymphocytes and determine the virulence of the virus.In recent years,experimental studies have shown that the native rabies virus glycoprotein(G)is a tiansmembrane glycoprotein in the form of a trimer.However,the immunogenicity and protective effects of soluble G protein monomers are not very rational,suggesting that the effective immunogenicity of rabies virus G protein is highly dependent on the integrity of the conformation of the G protein trimer.Fibritin foldon(Fd)is a T4 domain of phage fibritin protein,fibritin protein can help form a trimer,and through the salt bridge and hydrogen bonding and hydrophobic interaction approach to natural stable trimer structure.By using the properties of foldon,fusion proteins to form a trimer,and help to form the correct conformation of protein stability and immunogenicity of higher,so as to achieve better effect of treatment and prevention.At present,it has been widely used in influenza virus glycoproteins,HIV-1,RSV glycoproteins and so on,and has achieved satisfactory results.Since 1994,DNA vaccine has been proposed for the prevention of infectious diseases.Its feasibility has been seen in many animal models,so to be used for viral vaccine clinical trials.After more than 20 years of research,it has been proved that the DNA vaccine can cause effective cellular and humoral immunity to a variety of infectious diseases(viruses,bacteria and parasites)and noninfectious(allergic and neoplastic)diseases.At the same time,DNA vaccine has the advantages of low cost,simple design and management,relatively fast production,and new impetus for the development of DNA rabies vaccine.This study constructs a full-length G protein(mRVG)plasmid,G protein truncated(contain secretory signal peptide and extracellular domain,mRVGst)plasmid,G fusion protein(tRVGst-LI,tRVGst-IQG8)plasmid containing foldon domain,using eukaryotic VR1012 plasmids as vector.The recombinant plasmid can express G proteins of different sizes and immunize BALB/c mice with CpG as an adjuvant.After 3 times of immunization,the results showed that the mice produced a strong level of IgG.The mRVG antibody group IgG was 1.6 times the level of the mRVGst antibody group,and was 1.4 times that of the tRVGst-LI and tRVGst-IQG8 immune groups.The IgG antibodies secreted by the tRVGst-LI and tRVGst-IQG8 groups were 1.4 times that of the mRVGst immune group.After antibody typing and cytokine detection,the experimental results showed that the DNA vaccine produced a strong Th1 cell immune response.The activity of neutralizing antibody(RVNA)was detected by rapid fluorescent focus inhibition test(RFFIT),and the protective effect of DNA vaccine was studied by intracranial toxicity test.The results showed that the fusion antigen tRVGst-LI and tRVGst-IQG8 neutralizing antibody levels were 9.3 IU/ml and 12.7 IU/ml,and the protective force was 50%-67%.The immune effect was better than the truncated antigen mRVGst of 4.3 IU/ml,16.7%,but weaker than the full-length antigen mRVG of 17.4 IU/ml,with a protective force of 83.3%.And the results showed that the foldon domain could increase the immunogenicity of soluble fusion G protein.However,the immunogenicity of G protein is not only related to the extracellular region,but also to the transmembrane region and extracellular region.The immunogenicity of G protein is more dependent on structural integrity.