| Infectious bronchitis(IB) is an acute,highly contagious respiratory disease caused by infectious bronchitis virus(IBV).Nucleocapsid protein is highly conservativein evolution, its main functions are packaging nucleotide,synthesizing RNA of infectious bronchitis virus genome and activing cell-mediated immunity.Nucleocapsid protein contains a great quantity of antigen determinants,whose immunogenicity is only next to spike protein, may envoke a large amount of cross-protective antibodies against IBV.In this study N protein gene is cloned and expressed,and is used as a immunogen to develop polyclonal antibody,and using the antibody as a coating antibody,a sandwich ELISA for detecting infectious bronchitis virus antigen was developed,which may act as a useful tool to early diagnose and control infectious bronchitis.Infectious bronchitis virus appears more and more serotypes,new variants have been being found all the time,it is difficult to prevent and cure infectious bronchitis because of no or only part cross-protection among different serotype strains.In order to increase the IBV sIgA level in the mucosa surface of chicken nasal cavity,trachea and intestine and then obtain the aim of IB control,we attempted to make use of a strong mucosal immunization adjuvant Cholera toxin B subunit(rCTB),we used the mixture of N protein and rCTB or the fused expression product as immunogen to immunonize 7-day old meat chicken nasally.The main contents and research results as follow:1.Gene fused expression of nucleocapsid protein(np) of IBV and the Cholera toxin B subunit(ctb)After the ctb gene and np gene were fused,the fusion gene were expressed in Escherichia coli BL21(DE3) strain,the product was designated rCTB-NP.The SDS-PAGE results showed that the molecular weight of rCTB-NP proteins renaturated were about 72.0kDa and 92.0kDa respectively.Western blot showed N protein in rCTB-NP has strong immunoreactivity.GM1-ELISA confirmed CTB protein in rCTB-NP had good immunologic competence.2.Study on the mucosal immunization effect of nucleocapsid protein of IBV7-day old meat chickens were immunized with NP,NP+CTB,CTB-NP respectively. IgG-NP,IgA-NP in sera,sIgA-NP in nasal,trachea and intestinal mucous membrane were detected by ELISA.After the second immunization and third immunization,high level IgG-NP and low level IgA-NP in sera were detected.SIgA-NP level in nasal wash, trachea wash and gut wash were enhanced significantly by CTB(p＜0.05),rCTB-NP group or rCTB+rNP group are significant higher than difference rNP group(p＜0.05).The sIgA level in trachea wash solutions were rCTB-NP group＞rCTB+rNP group＞rNP group. In conclusion,when CTB was mixed with or fused expression with NP as a inasal immunogen,sIgA level in nasal,trachea and intestinal mucous membrane can be enhanced obviously.3.Development of sandwich enzyme-linked immunosobent assay for IBVA sandwich ELISA for detection of IBV antigen was developed by using purified chicken anti-NP IgG as coat antibody and rabbit anti-IBV IgG as the second antibody. The results showed that the optimal work concentration of anti-NP IgG and rabbits anti-IBV IgG were 2.5μg/mL and 5μg/mL respectively.The sensitivity of the method was eight times higher than hemagglutination test.No cross reaction was observed with Newcastle disease virus(NDV),avian influenza virus(AIV),eggs drop syndrome virus (EDS-76V) and infectious bursal disease virus(IBDV) which demonstrated the method has high specificity and sensitivity. |
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