Cloning And Sequence Analysis Of Full-length Segment A Of An Infectious Bursal Disease Virus (HB-bp) Strain

Sequence and phylogenetic analysis of segement A of Chinese infectious bursal disease virus (IBDV) HB-bp strain isolated from HeBei Province was studied in this thesis .The experiments as below were included: Double-strand RNA of viral genome was purified by Licl gradient precipitation ,48 hours after bursa was harvested from chicken which had been inoculated with HB-bp strain. Referred to the published sequence two primers were designed and synthesized . The full-length segment A of IBDV cDNA was amplified by RT-PCR and checked by agrose gel electrophoresis and purified by purified kit. The purified production was cloned into pMD18-T vector. The cloned plasmids were transformed into JM109. The Specific recombinant plasimid was identified by molecular weight ,PCR and restriction endonuclease analysis.The results indicated that the resultant construct contained the gene of IBDV A fragment at right orientation. The nucleotides sequences of the HB-bp strains vvIBDV segment A were determined.The results shows that the cloned 3142bp contains two overlapping ORFs.The large ORF of 3036bp coding a polyprotein(VP2-4-3),whereas the small ORF is 435bp coding VPS.The deduced amino acid sequences HB-bp strain were compared with those of other serotype I and serotype II strains.The results showed that the sequence of HB-bp segement A shared 95.4~98.8 homology with other serotype I at nucleotide level. It was very similar to vvIBDV from Europe, Hong kong ,Israel and Japan nor the variant strains from the United Stated and China Guang zhou. The specific sequence of vvIBDV were found from VP2, VP4 and VP3 regions.They were 222A,242I,256I,294I , 299S from VP2,680Y, 6851N, 714S from VP4,751D,990V,1005A from VP3 respectively. The result shows that a vvIBDV strain was obtained , The above work lay a important role for further studying on the molecular biological mechanism of antigenic drift and virulence variation of IBDV, molecular epedimiology, it also provided the basis for recombinant and gene deleted vaccine of IBDV.