Development Of Detection Methods For Rabies Virus Antibodies In Dogs And Cats

appear, there is no treatment and the disease is almost fatal. There have been three major rabiesepidemics in China since the 1950s. At present the third epidemic occurred. The peak of currentepidemic is yet to be reached. Facing the serious epidemic situation, inoculating main reservoirs dogsand cats is the key to preventing and controlling rabies. It requires effective methods to detect antibodiesagainst rabies in dogs and cats.Therefore, based on the molecular biological and modern immunological technique, we developeduniversal detection methods for antibodies against rabies virus in dogs and cats.The N gene of rabies virus Flury LEP strain was amplified by RT-PCR and cloned into pET-32a (+)expression vector. The recombinant plasmid pET-RV N was transformed into E.coli BL21(DE3).Thetransformed bacteria were induced by IPTG. SDS-PAGE analysis showed the recombinant RVnucleoprotein with a molecular mass of approximately 70 ku was expressed in inclusion bodies in E.coli.Western blot proved the recombinant RV nucleoprotein had strong immunoreactivity to rabies virusantibodies.Using purified recombinant RV nucleoprotein as coating antigen, an SPA-ELISA for the detectionof rabies virus antibodies in dogs and cats was developed (6μg/mL of recombinant RV nucleoprotein forcoating, the dilution of sera was 1:200, and the working concentration of SPA-HRP was 1:2000).Compared with a commercial ELISA kit coating rabies virus as antigen, the coincidence rate betweenthese assays was 96.67%.These results were suggested that this SPA-ELISA based on the recombinantnucleoprotein could be a useful assay to detect large numbers of samples and monitor RV antibodies indogs and cats.To develop a gold immunochromatographic assay for detection of rabies virus IgG antibodies, SPAwas conjugated with colloidal gold particles prepared by the tri-sodium citrate reduction method.Meanwhile recombinant rabies virus nucleoprotein and anti-SPA antibodies were striped in the test lineposition and in the control line position respectively. 93 dog serum samples were collected to evaluatecharacteristics of the gold immunochromatographic assay in comparison with existing commercialELISA kit. The coincidence rate of these two methods was 94.62%. Furthermore, the goldimmunochromatographic assay is rapid (15min) and easy to perform. It suggests that the goldimmunochromatographic assay is an acceptable alternative for use in field diagnosis and clinicallaboratories lack of specialized equipments.The G gene of rabies virus Flury LEP strain was amplified by RT-PCR. In Bac-to-Bac baculovirusexpression RV G gene was cloned into pFastBac HT A plasmid. The recombinant plasmid pFastBac-RVG was transformed into E.coli DH10Bac to generate the baculovirus shuttle vector bacmid-RV G byhomologous transposition and then transfected sf21 insect cells. PCR analysis indicated the recombinant baculovirus containing RV G gene had been constructed. Western blot demonstated RV glycoprotein wasexpressed in insect cells with a molecular mass of approximately 60 ku. Our results provide basis andproofs for further research.In general, the present results indicated that the purified recombinant RV nucleoprotein wasabtained by genetic engineering technology. SPA-ELISA and gold immunochromatographic assay weredeveloped using recombinant nucleoprotein as antigenic protein. The sensitivity and specificity of thesemethods are high. Our results lay the foundation for the production of RV antibodies detection kit fordogs and cats.