| Infectious bronchitis (IB), caused by infectious bronchitis virus (IBV), is an acute and highly contagious disease in chickens. IB has been an important disease of poultry around the world, and caused serious economic losses to the poultry industry. To monitor and study the genetic evolution of IBV strains epidemic in Sichuan province in recent years.,19 strain IBVs were isolated from some areas of Sichuan between 2008 and 2010, and the S1 genes of the isolations were cloned and sequenced. The sequences were compared with those of other reference strains in GenBank, and the genetic evolution was analyzed. Prokaryotic expression of the hypervariable region and high antigenic region of S1 glycoprotein of the delegate strain was also conducted. Followed were the main contents of this research:The lung and kidney of the diseased chickens from vaccinated chicken flocks in some regions of Sichuan province (including Chengdu, Meishan, and Leshan et, al.), were inoculated into the 9 to 11 days of non-immune chicken embryos for virus isolation and passage, and biological characterizations of the virus were investigated by hemagglutination test (HA), dwarfing embryonated chicken eggs and reverse transcription polymerase chain reaction (RT-PCR). The results show that 19 strains of IBV were isolated. A pair of primers were designed on the basis of published sequences of spike genes of IBV strains in the GenBank, the amplification segments were about 1,730bp encompassiong the entire S1 gene, that were amplified by RT-PCR. The fragments were ligated with a TA cloning vector pMD19-T and transforming into E.coil DH5a competent cell. Confirmation of clones containing recombinant plasmids were achieved by PCR and restriction enzyme digestion by BamHI and Hind III, and the recombinant plasmids were sequenced. Homology analysis of the nucleotide sequences and the deduced amino acid sequences of S1 genes of 19 isolates and 37 references strains chosen from GenBank were conducted by using the software of DNAStar, and Neighbor-Joining (NJ) tree was conducted with MEGA 4.0 software. The results showed that 19 isolates of nucleotide and deduced amino acid sequences homology were 71.7%-99.9% and 72.1%-99.6%, respectively, homology with reference strains were 70.8%-99.8% and 71.7%-99.4%, respectively. The 56 IBVs were separated into 8 distinct genotypes, and the 19 isolates were belonged in 4 different groups, fifteen isolates were included in genotype LX4 which is the predominant genotype in the field flocks in China, one was belonged to TW-I type which is the special genotype in Taiwan, one was belonged to the argumentative genotype "proventriculus-type", two were belonged to genotype Mass, most of which were vaccine strains. Alignment analysis results of deduced amino acid sequences of S1 glycoprotein showed that there were many mutations, insertions and deletions in IBV isolates of LX4-type when comparing with other genotype IBVs. Representative LX4-type IBV isolate Sczy3 was used for inoculation in non-immune chicken sat the age of 15 days for studying the pathogenicity. Clinical symptom was observed 3-15 days after challenge in 100% of chickens, and the mortality was 40%.According to the bioinformatics analysis result of the S1 gene of isolates Sczy3, the secondary structure of S1 glycoprotein were composed mainly ofβ-strain andβ-turn containing some irregular coil regions and littleα-helix regions and well immunogenicity. A strong antigenic hypervariable region located in 132-341 aa of S1 glycoprotein was chosen for prokaryotic expression. The nucleotide sequence was amplified and cloned into pET-32a (+) vector to generate recombinant plasmid pET-32-S1. The result of SDS-PAGE showed that recombinant protein about 43 kDa were successfully expressed Western blot analysis showed that the protein can react positively with rabbit hyperimmune serum against IBV.
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