The Research Of Suspension Array For The Detection Of Canine Rabies Virus Antibody

Rabies, which caused by neurological rabies virus, is a highly lethal zoonosis. It almost can infect all warm-blooded vertebrates and the mortality rate is nearly 100%.For the pet and ordinary experimental dogs, it needs to detect the rabies virus antibody levels in serum regularly, which can monitor and evaluate immune effects.Especially, the rabies antibody of SPF canine must be negative. Currently, the technologies for detecting the serum antibody levels of canine rabies virus cannot detect rabies and other common canine viral infectious diseases at the same time.Suspension array is a new type of high-throughput detection technology, it can simultaneously detect a variety of biological molecules with a sample. Compared with detecting a variety of pathogens or antibody respectively, suspension array can save considerable time and also greatly improve the utilization of the sample.The canine rabies virus glycoprotein artificial antigens were prepared in two ways with rabies virus glycoprotein expression and B cell antigen linear epitope prediction. The preparation of recombinant rabies virus glycoprotein antigen by means of insect baculovirus expression system: The glycoprotein gene amplified with RT-PCR was cloned into p MD18-T vector, recombinant cloning plasmid p MD18T-SRV9 G was successfully constructed after the identification of PCR and sequencing. The SRV9 G gene was cloned into p Fast Bac Dual plasmid again, and it was identified by PCR, enzyme digestion and sequencing, the results showed that recombinant transfer plasmid p Fast Bac Dual-SRV9 G was successfully constructed.The recombinant baculovirus transfer vector was transformed into DH10 Bac containing a shuttle vector of bacmid, and the colonies were identified by PCR analysis, the results showed that recombinant shuttle plasmid Bacmid-SRV9 G was successfully constructed. After Sf9 cells transfected by recombinant shuttle plasmid,the cell pathological changes were observed. When most of cells revealed to pathological changes, recombinant baculovirus was harvested and reproduced. then it was identified by the observation of negative-stain electron microscopy, the results suggested efficient gene transfer. By SDS-PAGE and Western blot experiments, it was proved that the recombinant baculovirus could express glycoprotein in insect cells. The prediction of rabies virus glycoprotein antigen epitopes: On the basic of glycoprotein basic properties and secondary structure analysis, eight B cell linear antigen epitopes with a high degree of probability were obtained by DNAStar,Bepipred and ABCpred. The sequences of screening were synthesized and used in the establishment of suspension array.This study expected to prepare capture beads through conjugated rabies virus glycoprotein and linear B cell epitopes TG16-1, TG16-2, GS-12, DG-14 on the carboxy-magnetic microspheres. Five kinds of capture besds were evaluated by detecting the same negative and positive sera. The RVG and epitope polypeptide TG16-1 were taken as detect antigens preliminarily. The epitope polypeptide TG16-1was coupled to magnetic fluorescent microspheres and polystyrene fluorescent microspheres to prepare capture beads and detect the same negative and positive serum. The results showed that polystyrene fluorescent microsphere was better than magnetic fluorescent microsphere. Two different types of capture microspheres were prepared by coupling RVG and TG16-1 to thiol polystyrene microspheres. The amount of capture microspheres, PE-labeled secondary antibody concentration,serum and PE-labeled secondary antibody incubation time were optimized. Both of the capture microspheres amount of RVG and TG16-1 were 1 μL, PE-labeled secondary antibody concentration 8 μg/m L and 4 μg/m L respectively, serum incubation time 2 h and 1 h, PE-labeled secondary antibody incubation time 1 h and0.5 h. ultimately, epitope polypeptide TG16-1 was taken as detect antigen because of easy synthesis, high purity, and be be beneficial to transformation for research achievements. The specificity and sensitivity of suspension array detecting canine rebies virus antibody were estimated. The results showed that it had no crossreaction with canine distemper virus positive serum, Canine Parvovirus positive serum or infectious canine hepatitis virus positive serum, and the detection limit of antibody levels was 0.41 IU/m L.The suspension array for the detection of canine rabies virus antibody has been successfully established in our study. The detecting technique has the advantages of strong specificity, high sensitivity. This method is the foundation for developing high-throughput rapid detection kit of major viral infectious diseases.