| Porcine reproductive and respiratory syndrome (PRRS) is a kind of infectious diseases characterized by breeding sows and piglets respiratory disorder, which caused by porcine reproductive and respiratory syndrome virus (PRRSV). The disease has attracted great attention around the world, the research has been started, but its pathogenesis is not clear. Some of the current study found that the antibody-dependent enhancement phenomenon during PRRSV, the PRRSV infection enhanced through the link of antibodies and target cells by Fc receptor on the cell surface, which may be related to the pathogenicity of the virus .In this test, The Hn/06 PRRSV were inoculated with the Marc-145 cells, virus fluid were collected after CPE, 20-day old piglets were intranasally infected Respectively, 10mL / head, on the 11 day, 21 day, 31 day after infection, The pigs were enhanced anti-viral immune with incomplete Freund's adjuvant containing purified concentrated virus, 5mL/head, The antibody titers were measured before each immunization, the anti-PRRSV pig blood sera prepared on 45 days after infection,and the antibody titers up to 1:12800 by ELISA.The healthy pig IgG were extracted using ammonium sulfate and DEAE-cellulose ion-exchange chromatography column, The rabbit anti-pig serum were preparated using the purified pig IgG, The antibody titers of rabbit anti-pig serum up to 1:64 by the agar diffusion test. Similarly, The rabbit anti-pig IgG were extracted with ammonium sulfate and DEAE-cellulose ion exchange chromatography, the protein was detected, The FITC fluorescence was labeled after concentrated at low temperature bag, the free of FITC molecules were removed from Sephadex chromatography after dialysis over 4h in PBS solution. The labeled rabbit anti-pig antibody were determination, the concentration was 6.99mg/mL.The Marc-145 cells were cultured in the 24-hole cell culture plate, the PRRSV were inoculated when it grow to more than 75%, The Marc-145 cells were fixed after 48h culture, with 80% cold acetone, washing with PBS solution, the Pig anti-PRRSV positive serum were diluted for 100,200,400,800,1600 and 3200 times for the first antibody respectively, setting the negative serum control. The FITC-rabbit anti-pig IgG were diluted for 10,20,30,40 and 50 times for the second antibody. According to square, the best work concentration of the first antibody and the second antibody were 1:800 and 1:20.200 TCID50 PRRSV and the diluted Pig anti-PRRSV positive serum Interact about 1h,which were diluted for 1:2,1:4,1:8,1:16,1:32, the PAM cells were cultured, After 48h, the PRRSV were detected by IFA, the Neutralization titer of Pig anti-PRRSV serum was 1:4.200 TCID50 PRRSV and the diluted Pig anti-PRRSV positive serum Interact about 1h,which were diluted for 1:8,1:16,1:32, made of different concentrations of antibodies and virus complexes, then,theComplex and the diluted guinea pig serum react on 1h ,which was diluted 10 times and had an equal volume. the PAM cells were Inoculated and cultured, After 48h, the PRRSV were detected by IFA. At the same time, the complexes and the diluted 10 times rabbit anti-pig IgG react on 1h,which were Constituted of different concentrations of sub-Neutralizing doses antibody and virus, the PAM cells were Inoculated and cultured, After 48 h, the PRRSV were detected by IFA. the PRRSV were inoculated to PAMs, as positive control. The results show that it did not occur the fluorescence as control experiment about complement receptor blocking and Fc receptors blocked trials. The complement and rabbit anti-pig IgG can successfully blocking of the complex about sub-Neutralizing doses antibody and the virus into the PAM cells.The PAM cells were cultured In the 24-hole cell culture plate, after 24 h,the mouse anti-pig FcγRⅡB receptor extracellular domain serum and mouse anti-FcγⅢreceptor extracellular domain and the mixed serum Respectively joined in the PAM cells,the PAMs were closed about 1 h and inoculated different concentrations of sub-dose antibody complexes with the virus, after 60h, the PRRSV in PAMs were detected with the IFA and RT-PCR. Also, a healthy mouse serum as negative control PAM cells, PAM cells were infected with PRRSV as the positive control. The results showed that mouse anti-pig FcγRⅡB can block the complexes into the PAM cells,that made of the virus and sub-antibody of serum diluted for1:8,1:16 and 1:32.But,mouse anti-FcγRⅢextracellular area serum can only blocked the complex into the PAM cells, made of virus and sub-Neutralizing of doses antibodies diluted for1:8.The mixture of diluted serum about two anti-Fc receptor extracellular region was able to block the complex into the PAM cells, made of virus and sub-Neutralizing doses of antibodies diluted for1:8,1:16.Receptor blocking test results show that the antigen-antibody immune complex can help PRRSV into the PAM cells in the course of PRRSV infection, when the antigen-antibody complex and the PRRSV exist in the same system. |
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