| Rabies is a zoonosis which symptoms for acute fatal encephalomyelitis caused by rabies virus (rabies virus,RV) infection of the central nervous system of warm-blooded animals and human, and almost all warm-blooded animals are easy to be infected,Once the disease breakout, its mortality rate is almost100%, that is one of the highest human fatality rate of acute infectious diseases, it has become the major epidemics of serious harm to public health. While there is no effective medicine for treating rabies except for mainly focusing on preexposure and postexposure immunization,so the effective diagnosis of pre-exposure and monitoring whether the effective protective antibody is produced after rabies vaccination or not,besides developing vaccine effectiveness assessment are very important.In order to construct fusion proteins that can generate agglutination and immune reaction,specific primers were used to amplify,from the recombinant plasmid pMD-G and pMD-2E8ScFv and with PCR,2E8gene and Kg gene, after which the two were spliced into a fusion gene by splicing overlap extension PCR method to construct prokaryotic expression plasmid pET-Trx-2E8Kg, which was then transformed into competent cell BL21(DE3)plysS. Finally, protein expression was induced with IPTG. SDS-PAGE analyses showed that the target fusion protein was successfully expressed and identified in inclusion bodies, with an expression accounting for23.5%of the total bacterial protein, whose molecular weight was77.7kD, exactly consistent with the expected size. The purity of the expressed protein reached98.9%after being purified by affinity chromatography and refolded by Glutathione reduction. Results of Western-blot detection and agglutination test on red blood cell indicated that the fusion protein2E8Kg has bifunctional characteristics,not only being able to have specific reaction with anti-rabies hyperimmune serum, but can combine with human red blood cell.With this bifunctional Fusion Protein as the detective antigen and the "O" type red blood cells as instruction cells,a method was established to quickly detect the G antibody to rabies virus. And titration test helped to single out the optimum antigen dilution, which was1:16, the optimum corresponding working concentration, which was106.26μg/mL, the best serum dilution, which was1:16, and the best duration time, which was25minutes. Tests on canine parvovirus(CPV) and canine distemper virus(CDV) with this developed method showed that there was no agglutination in positive serum while the standard serum of RV reacted strongly, which suggested that Rmg protein had very good antigenicity. Results of repeat detections of RV positive and negative sera with different batches of Fusion Protein turned out to be completely consistent, showing very good repeatability. Erythrocyte agglutination assay and fluorescent antibody virus neutralization test (FAVNT) were then repectively used to conduct clinical tests on1086copies of dog sera. Parallel comparison of the results discovered that the hemagglutination titer was equal to or higher than1:16(equivalent to neutralization titer0.5IU/mL in FAVNT test), suggesting that the immune antibody was qualified, and the coincidence rate of the two tests’results reached89.2%.To conclude, in this research, a diagnostic kit, used in erythrocyte agglutination assay to detect the antibody to RV, was developed by the Bifunctional Fusion Protein derived from2E8ScFv and Kg, having advantages of simple operation, high sensitivity and good specificity. It can be used to detect antibodies to dog rabies and to assess the immune effects of the vaccine, and is able to meet field demands of quickness, simplicity and on-the-spot application. |
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