| Reticuloendotheliosis virus(REV) is a kind of RNA virus that can lead to immune suppression and reticuloendotheliosis in infected chickens, and this virus infection was widely popular in chicken flocks in China. It brings great losses for poultry industry every year due to failure of vaccines caused by REV infection. It is regarded as a potential prevent measurement against REV infection using the inoculation of subunit vaccine or DNA vaccine. But it is very difficult to be widely used in clinic production because of their low protection for the inoculated chickens now; so how to improve the immnoprotection of these vaccines is a very important scientific subject by now. In this study, the ligands of Toll-like receptors, CpG ODN and Poly(I:C) were prepared as sustained release adjuvant, and were inoculated into chickens combining with REV subunit vaccine or DNA vaccine and their immunoprotection were evaluated in vivo and in vitro.Firstly, REV gp90 gene were amplified from REV-infected cells using the designed primers, and the recombinant plasimid containing gp90 gene was constructed and expressed in E.Coli cells, the expressed product was identified by western blot mediated by REV monoclonal antibody(MAb) and was used as protein antigen of REV subunit vaccine. Secondly, the recombinant eukaryotic plasimid containing gp90 gene(PVAX1-gp90) was constructed and transfected into chicken fibroblasts(CEF) and mammalian cells(BHK-21) with liposome 3000, the expressed product containing gp90 protein was identified with IFA mediated with REV MAb, and the plasimid was used as antigen of DNA vaccine. Thirdly, CpG ODN and Poly(I:C) were prepared as sustained release adjuvant and combined with protein antigen and DNA antigen, respectively. Two experiments were conducted:(1) The gp90 expressed products were inoculated into 7-day-age chickens combining with PBS, CpG-ODN or Poly(I:C) adjuvant, and two booster inoculation were conducted and then the chickens were challenged. The serum REV-antibodies was detected weekly after vaccination, and the viremias and the immunosuppressive effects caused by REV infection were monitored after being challenged. The neutralization effects of antisera were tested in vitro.(2) Recombinant DNA plasmids containing gp90 gene were inoculated into 7-day-age chickens combining with PBS, CpG or CpG+Poly(I:C) adjuvant and two booster inoculation were conducted and then the protection was avaluated as the previous.The results showed that:(1) the amplified gp90 gene contains 918 bp and could be expressed into 51 KD recombinant protein product, which can be recognized by monoclonal antibody(MAb)against gp90 protein. Compared with the chickens in gp90 group, the antibody responses(antibody titers and the antibody positive ratios) in CpG+gp90 group were greatly increased, the viremias and the inhibition on the antibodies responses to AIV/NDV vaccines caused by REV infection were significantly decreased. No significant increasing of antibody responses in Poly(I: C) +gp90 group, but the viremias and the inhibition on the antibodies responses to vaccines caused by REV infection were dramatically decreased.(2) Single CpG sustained releasing adjuvant and CpG+Poly(I: C) sustained releasing adjuvant can both improve the titers and positive ratios of REV antibodies to DNA vaccine, and can significantly reduce the viremia caused by REV challenge. In vitro, the results of neutralizing test show that the antisera with different titers from the inoculated chickens can inhibit the proliferation of REV in CEF, IFA results show that the antibody Ig in serum can directly bind to the virus particles in cells.Conclusion: CpG-ODN/Poly(I: C) combined with REV gp90 protein or recombinant DNA plasimids can both enhance the immunoprotection against REV infection, although the immune responses induced by them are very different. This study is very helpful to understand the immunoenhancement of CpG-ODN/Poly(I: C) and find new preventive reagent against REV infection in poultry production. |
PDF Full Text Request