Development And Application Of Rapid Detection Of Infectious Bovine Rhinotracheitis

Infectious bovine rhinotracheitis viru(sIBRV)can infect a number of tissue in the body. There are five different clinical syndromes due to infringing different tissue such as respiratory disease, conjunctivitis (pink eye), infectious pustular vulvovaginitis or balanoposthitis, abortion and encephalitis. Subsequent to primary IBRV infection, the virus establishes latent infection, the animal is then infected and shedding virus for entire life. The disease causes heavy economic losses among feedlot cattle industry in the world. It was classified as notifiable animal disease by World Organisation for Animal Health (OIE) and was listed as one of animal diseases of quarantine by Chinese authority. The viral genome encodes for 11 glycoproteins. The gB glycoprotein was one of the main protein in virus envelope surface displayed, it was also the major immunogens protein. It plays an important role in the process of pathogenesis and immunity. Five antigenic sites on the gB genome were found based on DNASTAR biology software analysis result of the IBRV Bartha Nu/67 strain gB gene sequences on GenBank published and the relevant information. The biological software Primer Premier 5.0 was used to design six pairs of primers. Two pairs of primers (outer primers and inner primer) for nested PCR amplification of total IBRV gB gene primers. The other four pairs of primers were for amplification of five antigenic sites. EcoRⅠand SalⅠsites were inserted in inner primers and the four pairs of primers of the amplification five antigenic sites. IBRV gB gene was connected to the cloning vector pMD18-T, named pMD18-T-gB. Five antigenic sites were amplified using recombinant pMD18-T-gB as a template. The fragments of five antigenic sites were connected to the pMD18-T cloning vector and expression vector pGEX-6p-1. The four recombinant expression vectors were constructed and transformed into E. coli BL21 (DE3) for expression. The SDS-PAGE electrophoresis result indicated the molecular weight of the four recombinant proteins were respectively 40.3kDa,39kDa,37.8kDa and 38.7kDa in concordance with prediction. Good reactivity of recombinant proteins was detected by indirect ELISA and Western blot. Furthermore the conditions of expression were optimized. The expression level was the highest at 1mmol/L isopropylβ-D-1-thiogalactopyranoside ( IPTG ) induction at 37℃for 3 hours. The ratio of recombinant protein were at 49%,45%,46%and 48%. The expressed protein was mainly present in cytorrhyctes observed by SDS-PAGE. Highly purified recombinant protein was purified by using GSTrap HP chromatography. The purity of gB3 was 95.27%.MDBK cell line was used to culture infectious bovine rhinotracheitis virus ( IBRV) Nu/67 strain. Cell culture supernatant was collected and purified using the sucrose density gradient centrifugation. Precipitated virus was determined its protein content and mixed with Freund's complete adjuvant and Freund incomplete adjuvant emulsification into the vaccine respectively. 4-6 weeks old female BALB/c mice were immunised with at the dose of 100μg per mouse every two weeks, the last time double dosage of immunization for the reason of strengthening. There are total 4 times for immunization. 3 days later, splenocytes were obtained and fused hybrid with SP2/0 myeloma cells using lymphocytes hybridoma technique. Two hybridoma cell strains, C5F4 and 3D5, were able to excrete stably monoclonal antibody. They were identified by using the method of indirect ELISA after cloning time after time. Specific monoclonal antibodies secreted by the two strains were verified to be IgG1 subclass, which showed good specificity and no cross-reaction with BVDV, AKAV, BEFV and IBAV. Antibody titers were measured by indirect ELISA. C5F4 monoclonal antibody titers were as follows: 1:1600 at supernatant, 1:12800 at ascites. 3D5 monoclonal antibody was determined after purification titer: 1:1280 at supernatant, 1:25600 at ascites. After continuously culture 20 generations the strains still secreted antibodies stably.Gold nanoparticles with good homogeneity and the average particle size of 22.7nm were prepared by nano-gold labeling technology, and the best pH value of colloidal gold solution determined was 7.5. The dosage of 30μg of monoclonal antibodies as a practical minimum amount of protein marker was confirmed by determination of the proportion of different concentrations of monoclonal antibody solution and colloidal. The TCID50 tirer of IBRV as to 10-7 was 10-fold progressive diluted from 10-1 to 10-8. 100μL only of sample was taken to drop the test strip well, after 10min, two red lines on the detection area and the quality control area appear, which showed that the result was positive and the sensitivity of the developed test strip for the 10-5, that is 100 TCID50. There is no cross reaction when the test strip inserted into the bovine viral diarrhea (BVD), Bovine Akabane Disease (AKA), bovine ephemeral fever (BEF), Ibaraki disease (IBA) virus samples. Ten known positive samples and ten known negative samples were detected with the strip, each sample repeated 5 times, the results were consistent. The test of stability showed that the strip was stable at the temperature of 4℃for two months. 246 clinical nasal swabs were collected and tested by the test strip, and compared with PCR, the negative consistent rate was 99.17% of two methods both.In this study, the method of gB3 blocking ELISA was established based on the recombinant IBR-gB3 protein test antigen, tested serum as a blocking antibody, a monoclonal antibody (3D5) as a detection antibody. ELISA working conditions were optimized: gB3 optimal coating concentration was 0.78μg/mL, the best blocking antibody dilution 1:15000, the optimal dilution of tested serum 1:20, working concentration of rabbit anti-mouse HRP secondary antibody at 1:15000. The results of to test 38 cattle sera positive samples by the newly created gB3 blocking ELISA showed that the sensitivity of this method is 97.37% and the specificity is 96.16%. It was proved that no cross reaction with bovine viral diarrhea (BVD), bovine Akabane disease (AKA), bovine ephemeral fever (BEF), Ibaraki disease (IBA) and bovine tuberculosis (BTB) positive sera. The plate variation coefficients were less than 10% proved by test of repeatability for the method. 1300 Breeding cattle sera from abroad were tested use imported kits and newly created gB3 blocking ELISA in the study. 1223 were found negative by use of established gB3 blocking ELISA compared to 1286 negative results by use of imported kit. The coincidence rate of two test kits was 95.10%.