Development Of ELISA Detection Method For Antibody And Colloidal Gold Immunochromatographic Strip Of Transmissible Gastroenteritis Virus

Transmissible gastroenteritis(TGE),caused by transmissible gastroenteritis virus(TGEV),is an acute and highly contagious disease in swine.Recently,TGE has been one of the main pathogens of diarrhea,and produced a great loss to the swine industry in Asian countries.The rapid and accurate diagnosis of TGEV is important for the prevention and control of the disease.However,there is no formal registrated TGEV detection reagents and kits for antigen or antibody.In order to control of the disease,it is necessary to develop a better diagnostic method.We conducted this study mainly including five parts as described below:1.Isolation and identification of TGEVThe feces of diarrheal piglets was detected by TGE Ag test kit and RT-PCR,the result demonstrated the sample is infected TGEV.A strain of TGEV was replicated serially in ST cell cultures,which could produce CPE in ST cell.The purity test indicated that no sterile,no mycoplasma and no extraneous virus were detected.The specification test manifested that the isolation could be neutralized by anti-TGEV antiserum and recognized by direct immunofluorescence antibody text.The complete genome of TGEV-NMG was sequenced,and the results showed that homology of complete genome is about 97.9%?99.9%.Phylogenetic analysis showed that this strain was more related to TGEV H165,H16,Miller,TS and JS2012 than to other reference starins.These proved the Isolated strain was TGEV,and named TGEV-NMG strain.2.Development of an indirect ELISA for detecting antibodies against TGEVAn indirect ELISA method was established with the concentrated and purified TGEV as the coated antigen after optimization of reaction conditions.The optimal virus dilution and antigen concentration was set at 1:2560 and 1.36 ?g/well.The coated time was 4?over night(10?16h).The sealing buffer was 5%skim milk and the sealing time was 2h at 37?.The serum samples dilution were set at 1:400 and incubated for 1h at 37? The dilution of the conjugate was defined as 1:20000 and the reaction time was 1h at 37? It was judged as positive when the cutoff value S/P?0.4,as negative when S/P<0.4.The experiments show that the method has good sensitivity,specificity,repeatability and coincidence rate,showing a better application prospects.3.The expression of N fusion proteinThe full-length N gene was cloned to prokaryotic expression vector pET-28a,and then recombinant plasmid was transformed into Rosetta(DE3).SDS-PAGE showed that the recombinant Ecoli.Rosetta could express N recombinant protein.The molecular weights of N recombinant proteins was 50kDa and the main expression form was soluble.The detection result of Western blot showed that N proteins can react with TGEV positive serum of swine.The result suggested that the expression of N recombinant protein had good antigenicity.4.Preparation of monoclonal antibody anti-N proteinPurified N recombinant protein was applied to immunize BALB/c mice to produce anti-N monoclonal antibody.And three monoclonal cell lines were produced by PEG-mediated fusion and screened by clonal culture,iELISA and IF A,which could steadily secrete anti-N monoclonal antibody were obtained successfully,named MAb-N-10#,MAb-N-17#,MAb-N-18#respectively.The subtype of antibody belong to IgGl.After 20 continuous passages,the supernatant could recognit TGEV by IFA,showing good genetic stability.Ascites was prepared and purified by affinity chromatography.The Mab showed excellent specificity and sensibility with TGEV through IF A.Preparation of monoclonal antibody against TGEV provides an important basis for the research of TGEV detection.5.Establishment of colloidal gold strip for detecting TGEVFor rapid detection of TGEV.20nm colloidal gold particle was made by the tri-sodium citrate reduction method,and tagging Mab-N-17#anti-N monoclonal antibody.Mab-N-10#anti-N monoclonal antibody and goat anti-mouse IgG were coated on test region and the control region respectively.The colloidal gold strip was developed according to the principle of colloidal gold immunochromatographic assay.The result showed the strip reacted with TGEV specifically and sensitive,which had a cut-off value of 102.1TCIDso/O.1mL.The accuracy of the strip was 100%compared to the RT-PCR and foreign company.