Complete Sequence Analysis Of Budgerigar Fledgling Disease Virus And Cloning And Expression Of VP1 Gene

Budgerigar Fledgling Disease (BFD) , caused by Budgerigar Fledgling Disease Virus (BFDV), was firstly reported in USA and Canada in 1981. Furthermore, BFDV was spread to Japan, Italy, Hungary, Germany and Australia. In 1994, BFD was reported in China and made great loss to bird-keepers. Now, BFD still emerges in china. Effective methods for disease prevention and control were still unfounded.There are three parts in the thesis. In the first part, the genome of HBYM02 strain isolated from Hubei province was sequenced and analyzed. The result suggested BFDV isolated from different host belonged to one gene type. By counting the number of mutated nucleotides, the fact was shown that the mutation rate of T antigen was the highest and agno protein was very conservative. Maybe, T antigen kept closed relation with the mechanism of the abroad range of BFDV host and agno protein made an important role in the transcription of late-phrase protein. A phylogenetic tree, based on the genomic nucleotide sequences of seven isolates, is presented. This tree shows three main branches. Taking into account of the relation of the hosts, the tree suggest a host-specific evolution of BFDV. On the other side, it was not possible to delineate a geographically based evolution, because the strains with high degree of relationship have their origins on different continents.In the second part, the major capsid protein VP1 gene was cloned into pMD18T vector correctly. The result showed that the VP1 gene was a highly conservative gene in BFDV genome.In the third part, the VP1 gene was expressed in E.coli expression system and Bac-to-Bac system separately. The results of SDS-PAGE indicated that the major structure protein VP1 gene, which was cloned in prokaryotic expressing vector pET32(+)a, was expressed in a highly level and the molecular weight of the recombinant fusion protein was about 58KD. Moreover, Bac-to-Bac baculovirus expression system was used to express VP1 gene, the molecular weight of the recombinant protein was about 42KD. The results of western-blot showed that protein expressed in two system had good reactivity to the BFDV positive serum.Briefly, we could know the evolution relation of BFDV from different host and the fact that isolates from different hosts belonged to one gene type by Sequencing and analyzing the complete sequence of HBYM02 strain, which is important for the research of molecular epidemiology of BFDV and different vaccines for BFDV. VP1 is the main structure protein and has the characteristic of immunogenicity. So, The expression of VP1 gene in prokaryotic expression system and eukaryotic expression system laid on foundation for the developmentof the diagnosis methods in serology and vaccines for BFDV.