| A new and specific strain of IBV causing proventriculitis was isolated from proventriculus and selected to study its biological characteristics and molecular biology.Ail structural protein genes(S 1.. S2~ M.~ N) were cloned by using the RT-PCR technique and then sequenced~. and analysed, All studies were listed below: l.A new kind of Disease occurred in the chick group in Qingdao area since Sep 1 996.The new virus isolate was propagated in the 9-11 -day-old chick embryo for 5-6 passages,and then several tests were done to identify the isolate, These tests include the test of returning virus to host; inirnuno-protectction test and using trachea ring culture to decide the titre of virus by viral neutralization test. All proved that the pathogen causing pathological lesion in proventriculus is a kind of avian coronavirus. Then this disease was temporally named as Infectious Bronchitis Proventiiculus Type, and this virus was designated as SD/97102(once called QXIBV) 2. A pair of primers were designed and synthesized according to the reported Si spike protein gene sequence of IBV. Then the viral RNA of QXIBV was amplified by reverse transcription-polymerase chain reaction(RT-PCR).The amplified product was analyzed by agarose gel electrophoresis, all appeared a fragment of 1657 bp as expected.The RT-PCR product was cloned into the pMDl 8-I vector ,and then the recombinant plasmid was sequenced.The result suggested that it was the SI spike gene 6 of IBV. The recombinant plasmid was designated pMDQXSI .The sequence has been published in Genbank,and the accession number is AF193423.Sequence analysis showed that the Si gene has low G+C contents at about 37.0%.There existed several RE cleavage sites such as Hind III, BamI-lI, B gill, Sac I and Sal I,but have no EcoR I site ,which is different from that of other strains. Its homology compared with other IBV strains was between 87.02% and 94.21%.In the site between 154 and 429 of this SI gene is a highly variation region. The SI protein is composed of 540 amino aicd residues after the gene was translated into amino acids.the p115 8.24.In the inner of the protein there exist 18 Cys residues.The sequence of the cleavage site between Si and S2 subunits is HRRRR,which is different from that of other IBV strains (RRFISRR). Three high conservative regions were located at 169-181,230-250.485-506 amino acids residues. 3. Two pairs of primers were designed to amp1if~?the upstream (1 lOObp)and downstream (900bp)sequences according to the reported IBV S2 gene sequence in Genbank. .The two sequences comprised the whole fragment of 52 subunit gene. All these two sequences were cloned into the pGEM I-easy vectors. The sequences were then analyzed using the software DNASTAR and BLAST. The result showed that the S2 gene was more conservative than Si gene .The amino acids located between 560-600 are probably associated with the envelope membrane. 4.A pair of primers were designed and synthesized according to the reported nucleocapsid protein(NP) gene sequence of IBV. Then the viral RNA of QXIBV was amplified by reverse transcription-polymerase chain reaction(RT-PCR).The amplified products were analyzed by agarose gel electrophoresis, all appeared a fragment of 1248 bp as expected.The RT-PCR products were cloned into the pGEM-T easy vector .The PCR products were digested with restriction endonuclease PstI,XbaI and HindIII,and the recombinant plasrnid was sequenced.The results suggested that it was the nucleoca...
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