| Classical swine fever (CSF) is a kind of high contact deadly infection diseases and is considered important infectious diseases of serious hazard and restricting the development of pig industry in our country. Early as1956, developed the most effective in the world of Hog choleralapinized virus successfully controlled a large-scale outbreak of swine fever in China, but has not been eradicated. Distributing popular swine fever in China in recent years, and its prevalence and clinical characteristics also undergone a lot of changes. In recent years, the swine fever epidemic in our country presented the new features of atypical chronic, persistent infection and latent infection, Immune swine fever of pigs happen more and more, the prevention and control of swine fever presented new challenges.The present stage, the method of antibody levels monitoring of vaccined pigs is ELISA, this method requires several hours to complete, and requires skilled experimental operating personnel and special equipment, can not meet the damand of monitoring antibody levels in the clinical fast, reliable, inexpensive. Therefore, there is a very important significance to establish a rapid detection method for CSFV antibodies.The studies have shown that the neutralizing antibodies anti-CSFV could be mainly induced by the envelope glycoprotein E2and is preferred antigen for the establishment of CSFV antibody detection methods. In this study, the colloidal gold immunochromatographic rapid test for detecting CSFV antibody was established by using prokaryotic expressed fusion E2protein of CSFV. The results were summarized as followThe primers were designed based on the genome sequence of E2protein gene of CSFV, and the E2gene fragments lacked the signal peptide and transmembrane region, about650bp, was amplified. The fragment was cloned into the prokaryotic expression vector pET-32a (+), and a high-efficient expression vector pET-32a-E2was successfully built. Then the vector was transformed into E. coli BL21(DE3), induced by IPTG, SDS-PAGE electrophoresis analysis initially identified restructuring a fusion protein expressed in the supernatant, the size is approximately38kDa. E2protein was purified by nickel column and Western blotting analysis showed that the fusion protein E2-pET32a has immunogenicity. Immune the New Zealand white rabbit with purified E2protein to prepare the rabbit anti-E2protein polyclonal antibody. The protein concentration of antiserum is2.1mg/ml after the coarse extraction and purification.A good quality colloidal gold particles were prepared by sodium citrate reduction method, its size was about30nm. The purified E2protein labeled with colloidal gold was chosen as the capture antigen, the purified E2protein and rabbit anti-E2IgG was coated on the detection and control lines of the nitrocellulose membrane (NC), respectively, to prepare the colloidal gold immunochromatographic detection test strip. Confirmed by the experiment, the optimal pH value of colloidal gold labeling protein, concentration of E2protein, protein coated liquid, coating concentration of E2protein, coating concentration of rabbit anti-E2IgG is7.5,9.6mg/ml,0.01mol/L pH7.2PB+0.1%BSA,1.5mg/ml,1.0mg/ml. The best form u la of block buffer is0.5%PVP+0.75%Tween-20+0.5%PEG20000+2%BSA+0.02%NaN3+10mM pH7.2PBS. The specific detection of the test strip shows that the strip has good specificity and don’t cross-react with antibodys of BVDV, PRRSV and PCV2. Diluted of CSFV standard antibodies to1:16can still find the bands, this suggests that the gold dipstick has good sensitivity.A total of80serum samples from Taihu lake and Pukou in Jiangsu were detected with the test strip and IDEXX swine fever antibody ELISA test kits, the two methods of coincidence rate was90.3%. |
PDF Full Text Request