Construction,immunogenicity and protective efficacy of the Spike glycoprotein(S1) of infectious bronchitis virus with a replica.pdf

1、Infectious bronchitis virus (IBV) a member of the Coronaviridae family causes tremendous economic losses associated with production inefficiencies and mortality in poultry industry worldwide. In recent years nephrotropic

2、 IBV has been a major problem in poultry particularly in China. In many cases, the renal damage was observed in IB-vaccinated flocks, which suggested that currently IBV vaccines might not providing adequate protection. I

3、n ovo vaccination remains an attractive option for a cost effective, uniform and mass application of vaccines for commercial poultry. However, the vaccines which can be delivered safely by this method are limited and the

4、re is no currently licensed embryo-safe vaccine against infectious bronchitis virus (IBV).The contents ofthe thesis contain four parts which are briefly described as following; l. Identification of a nephropathogenic str

5、ain of infectious bronchitis virus (IBV): Nine-day-old specific pathogen free (SPF) chicken embryos were inoculated with XDC-2 strain of infectious bronchitis virus (IBV). The virus titre, 50％ embyo infectious dose (EID5

6、0) and pathological lesions in the developing embryo were detected. In another experiment, 18-day-old SPF chickens were inoculated with the same strain ofIBV while 5 chickens were injected sterile PBS as control. The res

7、ults of the first experiment showed that the virus caused dwarfness and death ofthe embryo after 3 days of inoculation. The EID50 of the virus was calculated to be 5xl0<'-5.33>/mL. All the chickens inoculated with the

8、virus showed the typical IBV signs while some chickens were died showing kidney enlargement, nephropathy, and massive uric acid salt deposit. Histopathological examination revealed that the kidney had tubular ectasia,

9、glassy degeneration of epithelial cells, endothelial cell necrosis and exfoliation in some vessel lumens, monocytes infiltration, hyperemia and hemorrhage in the renal interstitium. The virus was reisolated and detected

10、from the kidney by RT-PCR_ It indicated the XDC-2 strain was a predominantly nephrotropic infectious bronchitis virus.2. Cloning, prokaryotic expression and identification of truncated Spike gene ofIBV (Sf200) and chicke

11、n granulocyte-macrophage colony stimulating factor (GM-CSF): The truncated S1 gene of nephrotropic infectious bronchitis virus (IBV) and granulocyte-monocyte colony stimulating factor (GM-CSF) of chicken were amplified a

12、nd cloned into pMD18-T vector. Truncated S1 gene designated as Sf200(containing five antigenic sites of S1 glycoprotein on amino acid residues (aa) 24-61, (aa)291-398 and (aa) 497-543 and GM-CSF were then amplified from

13、 the respective recombinant pMD18-T plasmids and cloned into pET-32a (+) vector resulting pET-Sf200, pET-GM which were identified by restriction enzyme digestion and sequencing analysis, The prokaryotic expression of tr

14、uncated Sf200 and GM-CSF constructs was performed. The proteins were separatedthrough SDS-PAGE which showed a molecular mass of approximately 40kDa and 29kDarespectively, Polyclonal antibodies were raised in SPF mice by

15、injecting E.coli expressed Sf200 and GM-CSF and were used to identify these recombinant proteins and later adenoviruses by western blot analysis. These findings indicated that the polyclonal antibodies expressed in mice

16、 can be used to detect the recombinant truncated Sf200 and GM-CSF and vice versa.3.Immunogenicity and protective efficacy of a replication-defective infectious bronchitis virus vaccine using an adenovirus vector and admi

17、nistered in ovo: In this experiment, a recombinant adenovirus expressing the S1 gene of nephropathogenic IBV(rAd-Sl) was constructed and the immune responses and protective efficacy against homologous challenge were eval

18、uated after in ovo vaccination. The results showed that the rAd-S1 led to dramatic augmentation of humoral and cellular responses in birds vaccinated in ovo followed by an intramuscular boost. Both IFN-y and IL-4 in chic

19、ken's lymphocytes were induced by this strategy. Following challenge with IBV, the chickens vaccinated with recombinant adenovirus showed fewer nephropathic lesions and less severe clinical signs as compared to those rec

20、eiving wild-type adenovirus or PBS. 4.Protective immune responses induced by in ovo immunization with recombinant adenoviruses expressing Spike (S1) glycoprotein ofinfectious bronchitis virus fused/co- administered with

21、granulocyte- macrophage colony stimulating factor: In this study, the recombinant adenoviruses expressing chicken granulocyte-macrophage colony stimulating factor (GM-CSF) and S1 gene ofnephropathogenic IBV were construc

22、ted and characterized. Then, the immunological efficacy and protection against homologous IBV challenge were assessed in specific pathogen free (SPF) chickens. The results showed that the chickens vaccinated in ovo with

23、rAd-S1, rAd-GM-Sl (GM-CSF fused with S1 using glycine linkers) and rAd-GM-CSF plus rAd-S1(co-administered) developed specific anti-IBV HI antibodies. Moreover, the fusion of the GM-CSF markedly increased spleen cell pro

24、liferation and IFN-γ production while mild increased in IL-4 production, which demonstrated the enhancement of cell-mediated immune responses. Following challenge with IBV, the chickens in the group vaccinated with rAd-S