| Infectious bronchitis (IB) is a highly contagious and acute disease of chickens. Now it has been spreaded to the whole world, and has posed substantial economic losses to the world poultry industry. Because the genome of IBV strains are continuously evolving by the frequent point mutations and genomes recombination, currently, dozens of serotypes and genotypes of IBV have been characterized, and cross-protection between different strains is nonexistent and weak. Therefore, it will provide great helps for IB prevention and control to do bioinformatics analysis of the genome structure of IBV.In this study,20pairs of primers were designed making reference of the gene sequence of IBV SC021202strain. The internal genomic sequence of IBV strain HH06were segmented amplified using RT-PCR, While the5’UTR and3’UTR non-coding regions of its genome were amplified using RACE kit, all amplified fragment was cloned and sequenced. The sequencing results were spliced together, its complete gene and each gene sequence were analyzed with bioinformatic tools of MEGA5.0and DNAStar. The results showed that the complete genome of HH06consisted of27,663nucleotides, and it has the typical characteristics of IBVgene sequence. Through the comparison of the whole genome and every gene sequencewith reference IBV, it was foundthat the length of3a,3b,5a,5b and N gene was most conservative, have the same size of sequence;5’UTR,3b, E and M genes are relatively conservative with moderate modification; insertions and deletions mainly occoured in la,1b, S gene and3’UTR region. Homology analysis showed, HH06strain and other strains genome nucleotide had homology from86%to96%, and had the highest nucleotide sequence identity to SC021202IBV strain, while had a lowest one to BJ IBV strain. Homology in5’-UTR, ORFlb, E, M,5a,5b and N genes with reference virus had a relatively high homology of99%-80%. Phylogenetic tree analysis showed that, HH06strain has closer genetic relationship with IBV SC021202strains and distant genetic relationship with the Mass-type strains. In the phylogenetic tree analysis of every gene, it showed big variation for different strains, and related to the conservation and variability status during evolution. According to the difference S1gene, all IBV strains were genotyped, the results show HH06strains belong to HN08-type. At the same time, S gene cleavage sites of the different IBV strains was also be analyzed, different serotype of IBV strains had not the same cleavage site and their amino acid positions were also showed different, the cleavage site of IBV HH06is536-540position of RRFRR. In addition, recombination and selection pressure analysis showed that HH06has a positive election and recombinant phenomenon.After antigenic and hydrophilic analysis of IBV HH06strains M protein, we removed its transmembrane fragment and was subcloned into prokaryotic expression vector pET-30a (+) and eukaryotic expression vector PVAX1. The recombinant plasmid of pET30a-M was transformed into E. coli Rosetta (DE3) and induced with IPTG, the molecular weight of recombinant M protein is20KDa. The recombinant M protein could be recognized by positive IBV antisera in western blot. Then the purified recombinant M protein was used as antigen for immunization of rabbit to prepare anti-M polyclonal antibody. Indirect ELISA showed that the titer of anti-M polyclonal antibody up to1:218, and western blot demonstrated it had high reactivity and specialty with recombinant M protein and IBV. Furthermore, IFA test demonstrated that this polyclonal antibody could react with BHK-21cells transfected with PVAX-M1plasmid and IBV-infected Vero cells. The pathogenic effect of Vero cells infected with IBV Beaudette strain could be inhibited by anti-M polyclonal antibody and the inhibition rate was up to25.9%. The rabbit anti-M protein polyclonal antibody obtained in this study laid a foundation for further functional research for M protein and detection of IBV. |
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