Expression And Identification Of The Nucleoprotein Gene Of Rabies Virus

Rabies is an acute, progressive and fatal disease caused by a highly neurotropic virus of the family Rhabdoviridae, genus Lyssavirus. The viral genome is a non-segmented negative-strand RNA. The fatality rate is almost 100% and there is no effective therapeutic drug until now. China is one of the most serious epidemic countries of rabies, the epidemic case is increasing rapidly, which formed the third pandemic in our country since 1950. So, it is very necessary to develop a safe, rapid and specific method for detection of rabies virus in order to improve the monitoring capacity of prevention and control of rabies.The viral genome is a non-segmented negative-strand RNA which is used to produce five monocistronic mRNAs encoding 5 structural proteins. The NP is one of the 5 structural proteins. NP is an important antigen with the most conserved epitopes which has the same specific antigen determinant in all the rabies virus group and common antigen determinant in related rabies virus. So NP is the optimized antigen for rabies detection. Nowadays, expressed NP of rabies virus and anti-NP McAb is commonly used in rabies detection in other countries. The Bac-to-Bac Baculovirus Expression System provides a rapid and efficient method to generate recombinant baculoviruses. This method is based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (bacmid) propagated in E.coli. Besides that, pFastBac contains an N-terminal 6×His tag for purification of recombinant fusion proteins using metal-chelating resin and a TEV protease cleavage site for removal of the 6×His tag following protein purification. So in this study, we used Bac-to-Bac Baculovirus Expression System to express CVS-11 NP, which can be further used to develop a safe, rapid and specific diagnostic reagent for detection of rabies.The N gene of RV CVS-11 strain amplified by RT-PCR was cloned into pGEM-T Easy cloning vector and sequenced analysis. Cutting pGEMT Easy-N cloning vector and pFastBac transfer vector by restriction enzyme BamHⅠand HindⅢand then showed correct ligating. The NP gene was cloned into pFastBac transfer vector and named pFastBac-N. The pFastBac-N was subsequently transferred into DH10Bac E.coli competent cells, which contained the baculovirus shutte vector (Bacmid) and the helper plasmd to generate a recombinant bacmid. To abtain the recombinant Bacmid-N triplication resistance, blue/white selection and repeat PCR detection were employed. Recombinant Baculovirus AcMNPV-N (P1 Viral stock) was obtained by transfecting the Bacmid-N into the insect cell line of Sf9 after 60 hours. And PCR results showed that successful recombination was happened. After continuous inoculation two generations, the titer of P3 Vriol stock can reach to 1.86×10~8 PFU/ml. The high-titer stock was used to infect Sf9 cells for large-scale expression of recombinant N protein. The cell contents and conditioned medium were identified by SDS-PAGE, Western blot and direct ELISA assay. Supernatants collected from cells infected by recombinant Baculovirus was used to test different serum antibodies of pre-exposure vaccinated people. The results showed here indicated that we had got the recombinant baculovirus expressing NP of RV, and the expressed N protein revealed good immunoreactivity with both mouse monoclonal and polyclonal antibodies to RV. Meanwhile, we confirmed that the NP protein was expressed in interior of infected Sf9 cells. And IFA results also showed a good level of serum antibodies.Above all, in this study, we had got the recombined Baculovirus of the rabies virus CVS-11 N gene successfully and a series of assays showed that the CVS-11 NP was expressed in interior of infected Sf9 cells. The expressed CVS-11 NP had a molecular weight of 50.5KDa, which was in consistent with that of NP of natural rabies viruses. The expressed CVS-11 NP had a good antigenicity and can be great useful to further study in the diagnostic reagent of rabies virus detection.