Identification And Variation Analysis Of M, N Gene Of IBV Shaanxi Isolate

Infectious bronchitis (Infectious Bronchitis,IB) caused by infectious bronchitis virus (Infectious Bronchitis Virus,IBV), which results in heavy economic losses to the commercial poultry industry, worldwide. The virus causes respiratory disease, reduced production performances, nephrosis and irreparable damage to oviduct leading to production of abnormal eggs. Immunization programmes now seem to be ineffective due to the extreme genetic variations of IBV. To date information on IBV local epidemic strains has been scare, although the disease is endemic and continues to have a considerable impact on China poultry industry. Our objectives were to character two IBV isolates from Shaanxi province. Four different parts of work have been finished as follows: 1. Two IBV isolates were collected from suspected flocks with respiratory infectionsymptoms. The viruses were indentified based on inoculated chick embryo, HA test, molecular biology tests and animal infection. The 2 IBV isolates showed high nephropahtogencity and infectivity to chicken embryo.2. A pair of IBV special primers was designed according to the nucleotide sequences of IBV M gene published in GenBank. M gene of IBV isolate SX-H09 and SX-H09 were amplified by RT-PCR and the PCR produces were sequenced. The results showed that the ORF of M gene of isolate SX-H09 contained 678 nucleotides, coding 225 amino acids, while the M of SX-W09 contained 670 nucleotides, coding 222 amino acids. The isolates shared 86.5%～99.6% and 84.5%～99.6% homology with the reference strains based on nucleotides and deduced amino acids, respectively. Insertions, mutations and deletions were found in M gene of the two isolate. Phylogenetic analysis revealed that the SX-H09 isolate belongs to the clusters together with DY07, while the SX-W09 isolate can't be classified into the clusters formed by the know strains.3. N gene of IBV SX-W09 strain was amplified and sequenced. Homology analysis of the isolate with the reference strains of IBV in GenBank revealed that the N genes of IBV SX-W09 shared 86.5%～89.9% nucleotide identity and 89.0%～94.1% deduced amino acids identity with the reference strains. 1230 bp nucleotides, encoding 409 amino acids of the N gene shared more mutation and heterologous with reference strains.4. The PCR product of N gene of SX-W09 was digested with BamHⅠand HindⅢ, and then the products were purified and connected with pET-32a (+) vector. The recombinant plasmids were transformed into E. Coli. BL21(DE3) and induced by IPTG. The purified N proteins were used to product polyclonal antibody in rabbits. Results show that we has successfully constructed a prokaryotic expression vector to expression N protein of IBV and prepared polyclone antibody with a titer of 1:12800 special for IBV N protein.Two IBV isolate were isolated and identified in this paper. Based on animal challenge results, the two IBV isolated showed high nephropahtogencity. M and N gene were sequenced and analyzed. N special polyclone antibody was prepared in rabbit based on prokaryotic expressed N protein. The study charactered two IBV isolated from Shaanxi province and may provide useful information for control the disease caused by IBV. .