Expression Of G And N Gene, Preparation Of Monoclonal Antibodies And Development Of An Immunochromatographic Test Strip Of Rabies

Rabies has been described as a disease with a great impact on public health. Rabies, caused by rabies virus (RV), is an acute, progressive and fatal zoonosis, and caused encephalomyelitis. The fatality rate is almost 100%. It is also a worldwide disease, about 50000-60000 people were died of Rabies every year in the world, and the most (about 90%) were in Asia and Africa. Human rabies, in our country, is very serious and the main reservoir and transmission host are dogs and cats (about 95%). Forcible immunization and immunologic surveillance, and detection of virus for suspect of animals in endemic areas are main preventive and controlling measures. So based on the molecular biological and modem immunological technique, we developed a new immunochromatographic rapid test method for detection rabies virus antibodies, while possessing both specificity and sensitivity.According to the genome sequence of RV reported in GenBank, two pairs of specific primers were designed and synthesized. Both glycoprotein (G) gene and nucleoprotein (N) gene of RV ERA strain were respectively amplified by RT-PCR, cloned, sequenced, and sequence analysis. The results demonstrated that the full length of G gene of ERA strain was 1 353bp, which encoded 524 amino acids. The initial 19 amino acids were signal peptide and sequential 505 amino acids were mature glycoprotein. The overall length of N gene was 1 353bp, which encoded 450 amino acids. The homologies of nucleic acid and deduced amino acid sequence of glycoprotein and nucleoprotein compared with the standard strains (for CVS, PV, SRV9, SAD-B19 ) were 89.1%-99.1% and 98.0%-99.6%, 89.7%-99.3% and 98.3%-99.6%, respectively. There are three potential N-glycosylation sites (Asn-X-Thr/Ser) in deduced G protein for ERA strain, but 4 for PV and HEP strains, 2 for CVS, Kelev and Mokola strains. The N-glycosylation site 333 was possessed for most strains of RV, but 319 was only possessed for most Street and Fixed strains. The analysis of hydrophobicity, Jameson-Wolf antigenic epitopes and surface probability plot of G and N proteins of ERA showed that it was as well as with CVS, PV and other strains. The analysis of BC epitope, Th epitopes, antigenic epitopes (III, IV) and phosphorylation site showed that it was also as well as with Fixed strains and some isolated strains reported in GenBank. So not only the ERA strain has a good immunogenicity, but the both recombinant N and G proteins have a good immunoreactivity.To establish the indirect ELISA for detection of RV antibodies based on recombinant nucleoprotein. The N gene was obtained from pMD-N by enzyme digestion and cloned into prokaryotic expression vector pET28a(+) to obtain the prokaryotically expressed plasmid pET-N. The target gene was then expressed in the E.coli BL21 (DE3) cells by inducted with IPTG and the expression was optimized with an induction time of 4 hours. The highest expression of the target protein was up to 56.8% of total bacterial proteins through the analysis of gel scanning, and the good immunoreactivity to rabies virus antibodies was proved by Western blot analysis. By using prokaryotically expressed gene product of N from rabies, the indirect ELISA assay for detection of rabies virus antibodies in canine serum was established after management of the optional working conditions. Sixty three sera samples from vaccinated dogs were used to evaluate the preclinical test of the indirect ELISA and compared with ELISA kit. The detection results obtained by both tests showed 93.6% agreement.To make use of the Bac-to-Bac(?) Baculovirus Expression System express the glycoprotein. The RV G gene was cloned into transfer vector pFastBacl to obtain pFastBacl-G and then transform into DH10Bac to generate a recombinant Bacmid-G by homologous recombination, triplication resistance and blue/white selection. Recombinant Baculovirus AcMNPV-G was obtained by transfect the bacmid-G into the insect cell line of sf9 that can be used for preliminary expression experiments. After the baculoviral stock is amplified and titered, this high-titer stock was used to infect sf9 cell for large-scale expression of the recombinant G protein. SDS-PAGE analysis showed that the highest expression level of G protein could be achieved at 72h post-infection, and the good immunoreactivity to RV antibodies was proved by Western blot analysis. The indirect immunofluorescent assay (IFA) using dog anti-RV serum could identify the sf9 cells expressing the G protein in cell, and this can develop a new method to detect the serum antibodies of RV.Prepared and identified of monoclonal antibodies (McAb) against RV. With brain suspension of suckling mice inoculated with RV and emulsified antigens of CFA and IFA as immunogen, and the developed indirect ELISA of RV and recombinant N and G proteins for selection, we obtained three hybridoma cell strains stably producing monoclonal antibodies against RV, named 3-2C7, 3-3H3 and 1-5D6, and the three strains McAb named 2C7, 3H3 and 5D6, respectively. 2C7 and3H3 are against RV nucleoprotein but on cross reaction between each other, and 5D6 is against RV glycoprotein. Immunoglobulin subtype experiments showed that 2C7, 3H3 and 5D6 were pertaining to IgG2b, IgG2a and IgG 1, respectively. Fluorescent antibody (FA) was prepared by the IgG that was purified by Caprylic acid-Ammonium sulfate and affinity chromatography from the ascites of 2C7 conjugated with FITC, and the good specificity of the McAb were confirmed by direct immunofluorescence assay based on the detection for the BHK21 infected RV. The working concentration of the FA is 1:400. The Fluorescent antibody test (FAT) was developed based on the FA could be used for detection rabies virus antigen.To develop an immunochromatographic strip (ICS) for the serological detection of RV antibodies in serum based on colloidal gold immunochromatographic assay (GICA). In the strip, the goat anti-canine IgG labeled with colloidal gold was used as the detector, and the purified RV and Rabbit anti-goat IgG were blotted on the nitrocellulose membrane for the test and control lines, respectively. The evaluation of specificity and sensitivity of the ICS and comparing with FAVNT were performed by detecting the 63 clinical serum samples of immunized dogs, 40 serum samples from dogs infected with Canine adenovirus (CAV), Canine coronavirus (CCV), Canine distemper virus (CDV), Canine parvovirus (CPV) and Canine parainfluenza virus (CPIV) other than RV and 10 serum samples of RV free healthy dogs. The results showed that the strip has a high specificity and sensitivity that were closely correlated with those of FAVNT and the dependability of ICS with FAVNT is 94.0%. The lower detection limit of the ICS with naked eyes was 0.5～0.6IU/mL. Furthermore, the strip is rapid (8～12min) and easy to perform with no requirement of special skill, reagents or equipment. This suggests the ICS is an acceptable alternative to be used in clinical laboratories lacking specialized equipment as well as for field diagnosis.