Sequence Analysis, Prokaryotic Expression And Identification Of M Gene And Two Truncated M Gene Of Avian Infectious Bronchitis Virus (793/B)

Infectious Bronchitis(IB) is a severe acute respiratory disease of poultry, caused by Infectious Bronchitis Virus (IBV). Since broke out in USA in the 1930s, IB have been an important disease of poultry around the world. IBV is an sRNA virus, the gene of the virus always mutated owing to it's point-mutation and gene recombination, so the virus have many serotypes. The known serotypes can be classified into respiratory symptomatic serotypes such as Conn, Iowa97, JMK, Florida, Arkansas99 and nephritic symptomatic serotypes such as M41, Holte, Gray, Australia'T'.Gough reported a new serotype of IBV named 793/B (also known as 4/91 and CR88) in UK in 1992, 793/B isolates have not any or a little serological relation with other serotypes of IBV, the whole S1 gene of 793/B isolates differed by 21% to 25% from those of other 17 European isolates. In recent years, the 793/B serotype of IBV, harmful to the healthy development of pouLtry breeding, prevailed and spread in Spain, Germany, Holland, Italy and Thailand, too.Youxiang Diao, Jiehua Yang et.al.(2003) isolated an IBV 793/B strain named TA03 from layer flock in a chicken house of Shandong province. On the basis, an IBV 793/B RT-PCR detective method was established and S1 gene of IBV 793/B TA03 strain was cloned and expressed successfully by Shenye Yu et.al.(2007). Sequence of N gene of IBV 793/B TA03 strain was analyzed and Digoxigenin-labeled probe and multiplex RT-PCR for detection of the IBV were established and applied by Fanlei Meng et.al.(2007).According to the genomic sequences of IBV published in Genbank , a pair of primers was designed for amplifying the M gene of 793/B serotype of IBV by RT-PCR. The result of sequence analysis showed that the whole M gene of IBV isolated consisted of 669 nucleotides. Compared with 15 IBV strains which were published in GenBank, 3~12 nucleotides were absent in 4~15 position (1~4 corresponding aminoacids absence) and about 30 point mutations were found in the M gene. The homology of nucleotide sequences was 84.2%~93.6%, and the homology of deduced aminoacids was 82.1%~96.0%. Evolution analysis indicated that the TA03 strain was closely related to H52 and IBN strains. The results provided theoretical foundation for further research on the function of M gene (protein) in the genetic variation and immunization.To infer the antigen site of variant Avian Infectious Bronchitis Virus (793/B) M gene by bioinformatics technology. The primers of the M gene and the two epitope fragments were designed with DNAStar, and these fragments amplified by PCR were cloned into prokaryotic expression plasmid to construct the pGEX6p-M, pGEX6p-M1 and pGEX6p-M2, respectively. The expressed recombinant proteins GST-M, GST-M1 and GST-M2 can be detected at bands of 50 kDa, 32 kDa and 29 kDa by SDS-PAGE, and the results of Western blot confirmed that GST-M and GST-M1 not GST-M2 recombinant proteins could specifically react with the serum samples of mouse anti-IBV (793/B) respectively. The results showed that GST-M and GST-M1 recombinant proteins possessed a good antigenicity which initially confirmed the existence of the M protein epitopes. The study also laid an important foundation for further study on immunogenicity of 793/ B (IBV) M protein and preparation of genetically engineering vaccines.