Study Of 5a, 5b And N Gene Of Infectious Bronchitis Virus LX4 Isolated From China

Infectious bronchitis(IB) is an acute contagious disease caused by avian infectious bronchitis virus(IBV).In China, the study of IBV molecular biology is mainly focused on S gene, while the other genes are comparatively less. Thus, We further sequenced and analyzed 5a, 5b and N gene of IBV LX4, and expressed N gene in prokaryotic system after cloning and genetic analysis of IBV LX4 S gene.According to the published IBV gene sequence, a pair of specific primers were designed and synthesized. A fragment of 1.7kb including mRNA5 and mRNA6 cDNA of IBV LX4 was amplified, cloned and sequenced. Then the nucleotide and the deduced amino acid sequence of this fragment were analyzed. The results showed that the nucleotide sequence and deduced amino acid identity of LX4 5a gene have comparatively less identical with Beau, CU-T2, DE072, D1466, D971 and SD/97/02 strains, while the others identity is higher than 90%, while 5b gene shows high identical with reference strains. The result of nucleotide sequence and deduced amino acid identical comparison of N gene with Ark99, Beau, Gray, M41, HI 20, H52, SD/97/02, D971, ZJ971, DB, HB and N shows that LX4 was closest with HB and SD/97/02. The deduced amino acid sequence identity of three basic regions and other functional regions further proved that 5a, 5b and N gene of IBV LX4 shared more characteristics with other isolates from China than the aboard. In addition, the relationship between LX4 and the two vaccine IBV strains, namely H52 and HI20, was also found in N gene and its functional regions.The N gene of IBV LX4 was subcloned into pPROEX-Ta vector to construct recombinant plasmid pPROEX橦Ta-N and expressed in E. coli DH5a with IPTG induction. It was demonstrated by SDS-PAGE and Western blot that the two proteins, 56KDa and 45KDa, were expressed. The protein of 56KDa was close in size to native N protein and the other appeared to be the degradation product of N protein. The 56KDa protein was purified and used to prepare antibody against IBV N protein. The result showed that the antibody could react with different IBV, especially with its related strain, so recombinant N protein is one of suitable sources of antigen for IBV detection in chicks.