Prokaryotic Expression Of IBV N Protein And Development Of Indirect IBV N Protein-mediated ELISA

Avian infectious bronchitis is an acute and high contagious viral disease of chickens which is caused by infectious bronchitis virus. N protein is the most conservative one in the evolution of IBV, and of which is the major structural protein to determine the antigenicity of IBV. N protein can be involved in the binding of the RNA of virus, and induces humoral and cellular immune. Therefore, the study of biological function and characteristics of N protein is great significance for the diagnosis and prevention of IB. By expressing the highly conserved N protein, and using it as coating antigen to establish the indirect ELISA protocol which provide reliable theoretical basis and effective technological support for IBV antibody monitoring and disease diagnosis.In the study, according to IBV gene sequences published in GenBank, specific primers were designed to clone complete open reading frame of N gene by RT-PCR, an approximately1.75kb fragments of N gene was amplified and cloned into the pMD18-T vector. After N gene was proved successfully amplified by sequencing, we designed a pair of sub-cloned primers according to the N gene sequences we acquired, and an approximately1.23kb target fragment was amplified by PCR. Then this gene was inserted into pET-30a(+) vector resulting in a prokaryotic expression plasma pET-30a-N. After restriction-enzyme analysis and sequencing, the recombinant plasmid pET-30a-N was transformed into the E.Coli Rossetta, and induced with IPTG. We obtained approximately51kDa soluble protein. The results of SDS-PAGE and Western-blot analysis showed that the recombinant protein was expressed successfully and had well reactivity with IBV positive serum.The pET-30a-N recombinant protein purified with Ni agarose column was used as coating antigen to establish and optimize the indirect ELISA protocol. The results shows that the optimal dilution of antigen and serum concentration were2.5μg·mL-1and1:40, the optimal of blocking solutions was1%BSA, the optimal time of conjugate was2h, the optimal dilution of secondary antibody was1:1000, the optimal reaction time of substate was10min. The results of repeatability test and specificity test showed that the indirect ELISA method had good reproducibility and specificity, and has no cross reactions with other reference viral and bacterial disease positive serum. Furthermore,135serum samples from2chicken farms were assayed by this indirect ELISA, and got nearly the same results as those of IBV-mediated ELISA. It will provide a good tool for rapid diagnosis and epidemiological study of avian infectious bronchitis.