Study On Molecular Genetic Variation Of N Gene Of IBV Isolates From Shannxi Province

Infectious bronchitis (IB) is an acute, highly contagious viral respiratory disease of chickens caused by Infectious Bronchitis Virus (IBV). IBV circulates in chickens worldwide and induces serious economic consequences in poultry industry. Vaccination is the most efficacious measures to prevent this disease. However, IBV has a lot of serotypes, and its genome evolves continually. this makes the control of IBV more difficult,and vaccination invalid occurs frequently. N gene of IBV which codes the N protein has significant effects in the Viral mRNA's transcription,virus particle's assembled and it can induce the humoral- mediated immunity and cell-mediated immunity response. In this paper, the molecular genetic variation of N gene of IBV isolates from shaanxi province was studied to illuminate the mechanisms of vaccination inefficacy on molecular level. Meanwhile, the N protein of W118 was expressed and its reactionogenicity was detected.1. N genes of 8 nephrotropic IBV isolates (WH, YX, YL, BJ, WG, G5, FF2, B01) were amplified by reverse transcription- polymerase chain reaction (RT-PCR). The PCR products were cloned into pMD18-T vector and sequenced. Homology comparison and phylogenetic analysis were carried out among these isolates and the reference strains of IBV in GenBank using DNAStar software. Phylogenetically, the variability of N gene of 4 isolates was conspicuous comparing with reference strains(85.5%~87.3%). Deduced amino acids sequence indicated mutation of N protein mainly displayed sporadic point mutation. Mutational site did not show obvious regularity and no obvious dependablity with tissue tropism of IBV. The phylogenetic analysis indicated that the N gene of WH,YX,YL strain closed to that of the reference strain Ark99 and Gray which were in the same branch of phylogenetic tree. However, BJ,WG,G strains constructed a alone branch of phylogenetic tree. B01 strain closed to IBV strains M41,Beaudette and EP3 in evolution. The N protein of FF strain shared a high divergence compared with other strains. It demonstrated that the potential tendency of variability of IBV N gene.2. G5(W118) has become one strain of IBV polyvalency inactivated vaccine of Yangling lvfang biotechnology Ltd. To reveal genetic background and immune effect of W118 strain in the molecular level, S1,M and N genes of IBV W118 strain were amplified by RT-PCR. The products were cloned into pMD18-T and sequenced. S1,M and N genes were analyzed among the W118 strain and the reference strains in GenBank. The results showed that W118 S1 gene shared a nucleotide identity of 71.0% to 85.3% and an amino acid identity of 72.7% to 83.9% with 14 reference strains, M gene shared a nucleotide identity of 86.6% to 98.1% and an amino acid identity of 89.8% to 98.2% with 12 reference strains, N gene shared a nucleotide identity of 84.1% to 95.7% and an amino acid identity of 88.9% to 96.1% with 14 reference strains. According to the deduced amino acid sequence analysis, S1 protein included 19 potential glycosylation sites, 17 Cys and the cleavage site sequence Arg-Arg-Ser-Arg-Arg (RRSRR). There had one aa deleting in 25aa site, seven aa inserting into 73aa site, and multiples of special point mutated on S1 protein of W118 strain compared to H52,H120 vaccine strains. There appeared point mutation on 61aa, 204aa, 222aa sites of M protein, Five point mutations were found in the antigen region 175~241aa of N protein. The variation of W118 isolate strain related to deleting, inserting and point mutations on S1,M and N gene revealed the molecular mechanism of low protection of IBV live vaccine(H120, H52, W93).3. The secondary structure of N gene of W118 strain was deducted by bioinformation software and the antigenicity was analyzed. The results showed that N protein is easier to bind with RNA because of its composition of amino acid and characteristic of secondary structure. The antigenicity of N protein is superiority because of its multitude B-Cell epi-position . The N gene's prokaryotic expression vector N-pET32a of W118 strain was constructed. The positive clone was selected and the expression product of N gene induced by IPTG was detected by SDS-PAGE, while the expression protein reactionogenicity was detected by Western-blotting. It concluded that N protein can be used as diagnostic antigen of IBV because of its superordinary antigenicity.4. A pair of primers were designed and synthesized according to the sequences of IBV N gene in GenBank. The N gene of IBV W118 strain amplified by PCR and was cloned into pMD18-T vector, and the recombinant DNA vaccine plasmid of W118N-pSCA1 was constructed. The sequencing result showed that the recombinant DNA vaccine plasmid contained the correct orientation and sequence of N gene. This could provide a foundation for the further research of combination applications of IBV N gene DNA vaccine with S1 gene DNA vaccine including inactivated virus to substitude the Live attenuated IBV strains.This research showed that the N gene of 8 IBV isolates have had some degree variation, and parts of antigen region of N gene had a notable divergence comparing to the vaccine strain H120 and H52 in China. This may indicate one of the most important reasons for vaccine immunity failure of IBV on the molecular level. The sequence analysis of main structural gene(especially in S1gene)of W118 strain indicated the molecular mechanism that W118 strain as the inactivated vaccine could prevent the infection of epidemic virulent strain, and supposed the ultimate defect of live attenuated vaccines H120,H52 and W93 in immunity prevention. This research provided theoretical support for new engineering vaccine exploitation and enriched molecular epidemiology information of IBV in our country.