The Prokaryotic Expression Of Nucleoprotein Gene And Membrane Gene Of Infectious Bronchitis Virus And Development Of Indirect ELISA By Nucleoprotein

Avian infectious bronchitis virus is a member of Coronavirus genus. IBV is a single-strand positive-sense RNA genome. N protein is the most conservative one in evolution of IBV of which the main function is packaging nucleotide, duplicating RNA of IBV genome and activing cell-mediated immunity, contains a large number of epitopes, this protein of immunogenicity is inferior to the S protein, can induce a large number of antibodies. M proteins and their genes is relatively conservative in evolution. M protein is related with duplicating RNA of IBV genome, while M protein may be an important immunogen genes. Although IBV immunity for the majority of studies have focused on the S1 protein, but the other IBV structural proteins, such as the N protein, M protein is more conservative than S1 protein, so the research N protein and M protein for understanding the infectious bronchitis diagnosis and control inflammation have important practical significance. In view of above, an ELISA method was developed with the conservative recombinated N protein expressed in E.coli Rossetta that provide dependable and available technological safeguard for controlling of IBV .And in the same time M gene charactered serology was cloned from genome of IBV and M protein was expressed in E.coli Rossetta to contribute to study the immunogenicity of M protein.According to pubilished N protein gene and M protein gene sequence of IBV in Genebank, specific primers were designed and synthesized.An approximately 1.23kb fragments of N gene and 678bp fragments of M gene were amplified from RNA of the strain M41 of Infectious Bronchitis virus preservatived in our lab by RT-PCR and cloned into the pMD18-T vector. The recombinant plasmid pMD18-T-N and pMD18-T-M was proved to be successful construction by restriction -enzyme analysis and sequenced. Then pMD18-T-N and pMD18-T-M with BamHⅠand HindⅢrestriction enzyme digestion, the digested N-genetic and M gene fragments were cloned into the prokaryotic expression vector pET-32a, respectively transformed into the E. coli Rossetta, respectively induced with IPTG to carry out SDS-PAGE electrophoresis and Western blot detection.The SDS-PAGE electrophoresis and Western-blotting detection confirmed: the gene had been expressed solubly in recombinant pET32a-N(R)cells,the size of the N being approximately 63kD and 55kD, and The expression product could specially react to the anti-sera of IBV which indicating the expression of N have a good immunological activity; the gene had been expressed solubly in recombinant pET32a-M(R)cells, the size of the M being approximately 30 kD, and The expression product could specially react to the anti-sera of IBV which indicating the expression of M have a good immunological activity.After the experiment of N gene's prokaryotic expression, The pET32a-N fusion protein purified with Ni agarose column was used to establish an indirect ELISA method for the detection of antibodies againt IBV by selecting the conditions with the optimal dilution of antigen of N protein to be 1.80 ug/ml, the optimal Dilution of primary antibody(serum sample) to be 1:80 and the optimal dilution of secondary antibody (HRP-labeled goat anti-chicken serum)to be 1:5000. Following the optimal conditions of ELISA, the threshold value of ELISA was 0.351 ( X +3×SD=0.18+3×0.057). In the analysis of indirect ELISA cross specificity, it revealed a negative reaction with positive sera of Newcastle disease(ND), Avian influenza(AI), Infectious bursal disease (IBD), Egg drop syndrome (EDS76), serum of avian uninfected with Infectious bronchitis virus (IBV))and a positive reaction with standard positive serum of or from infectious bronchitis. In order to know its reliability, a repetitive experiment was conducted in different ELISA in the same time and in the different time that result in less than 8% variable coefficient of absoprotion. To evaluate the specificity and sensitivity of this ELISA, 80 field sera samples were collected from chickens. Compared IDEXX kit and ELISA methods that established in this study were consistent with experiment, the results of positive consistent rate was 92.3%, negative coincidence rate was 97.1%, the result show that the ELISA method does not appear false positive, indicating that this ELISA method has a certain reliability.Then a total of 80 chicken sera samples were axamined that were sent from several field farms. The results above indicated the assay to be a good method with strong specificity, high sensitivity and excellent coincidence and it could be used to fastly accnrately detect the antibodies aeainst IBV in the field sera samples.