The Construction And Verification Of Recombinant Rabies Virus Expressing TenascinC-FBG

The rabies is an acute zoonotic disease which is caused by the rabies virus that causes central nerve damage.And nearly 59,000 people worldwide die of rabies every year.China is one of the high incidences of rabies.The mortality of rabies is extremely high with nearly100% deaths.There is no effective treatment of rabies,and vaccination is the most efficient way to prevent rabies now.Rabies virus consists of 5 kinds of structural proteins and single negative stranded RNA.Glycoprotein,located on the surface of the cell membrane,is the only protein so far to induce neutralizing antibodies(VNA)in the body.Some studies have shown that FBG domain in Tenascin C can bind and activate TLR4.And some previous researches in this laboratory have found that TLR4 adjuvant can significantly improve the immune effect of rabies vaccine,so we assume that Tenascin C can be the adjuvant of rabies vaccine.In order to explore the effect of Tenascin C-FBG's structural domain on the immune response of the body that infected by the rabies virus,we constructed the recombinant rabies virus LBNSE-FBG-C through reverse genetic manipulation,which expresses the Tenascin CFBG domain,and studied the basic characteristics of the recombinant virus inside and outside the body.First,the FBG domain of Tenascin C protein is inserted in the pseudogene position between the G and L proteins of the parental rabies virus LBNSE which is preserved in this laboratory by a reverse genetic operating system,we constructed recombinant RABV expressing Tenascin C-FBG through reverse genetic manipulation,named LBNSE-FBG-C.The Western Blot proved the in vitro expression of the inserted gene,and drew the multistep growth curve of the recombinant virus plotted,respectively,which was almost consistent with the parental virus LBNSE,so it proved that the insertion of Tenascin C-FBG had no significant effect on the proliferation of rabies virus.In terms of the pathogenicity of the virus,the mice were infected by the intracerebral route,which showed no obvious clinical symptoms,and the body weight basically have an upward tendency.The result demonstrates that the expression of Tenascinc-FBG had little influence on the viral pathogenicity.To further study the Tenascin C-FBG domain has been reported in the literature to activate the TLR4 pathway.Therefore,we isolated the bone marrow-derived DCs of mice,detecting the inflammatory factors of downstream of the TLR4 pathway by using fluorescent quantitative PCR and verifying TLR4 pathway in DC cells,activated in vitro by the recombinant virus LBNSE-FBG-C.Then we studied the effect of recombinant rabies virus as an inactivated vaccine in mice and found that LBNSE-FBG-C produced higher neutralizing antibodies than LBNSE.In the case of studying the effect of the pathogenicity by inserting foreign genes in rabies virus of adult mice,we injected ICR mice with DMEM,LBNSE,LBNSE-FBG-C,and the result is that all three groups of mice had no obvious clinical symptoms and their weight all keep rising.In addition,the survival rate(90%)of mice that were immunized with the recombinant virus LBNSE-FBG-C was significantly higher than that of the LBNSE group(50%).In summary,LBNSE-FBG-C has little effect on the in vitro growth characteristics of rabies virus,can activate the TLR4 pathway,and can quickly improve the level of body's neutralizing antibody and the ability to protect body from rabies virus.Therefore,LBNSEFBG-C can be used as a candidate for a more effective inactivated vaccine.