Sequence Analysis And Expression Of DuCV ORF3 Gene

Duck circovirus (DuCV) was listed as a tentative member of the Genus circovirus. DuCV can do harm to duck aquaculture because of immuno suppression of ducks caused by DuCV. In this study, DuCV was classified according to the length of its ORF3 gene. And function of ORF3 was studied by prokaryotic and eukaryotic organisms.1. Cloning and sequencing and sequence analysis of DuCV ORF3In order to elucidate the ORF3 gene variations of different Duck Circovirus (DuCV) isolates and analyze their homology, the ORF3 gene sequences of 10 DuCV isolates have been obtained from the clinical tissue samples. Being analyzed with the known gene sequence, the ORF3 gene has two genotypes. The lengths of the two genotypes are 237bp and 297bp respectively. The nucleotide homology of ORF3 gene in the same genotype is between 95.8-100% and it is only 87.8-91.6% between the different genotypes. Furthermore, the amino acid homology of this gene in the same genotype is between 88.6-100% and it is only 77.2-81.0% between the different genotypes.2. Prokaryotic expression of FJ0601 DuCV ORF3Incomplete ORF3 gene of DuCV II was obtained by polymerase chain reaction (PCR) and then expressed in prokaryotic organism. Purified protein was used for mice immunization to make serum. Then, antibody specific to protein of ORF3 was detected in positive serum of DuCV by ELISA. Results indicate the ORF3 gene has already been expressed in the duck and protein of it stimulated the production of corresponding antibody. Based on this, it can be inferred that ORF3 of DuCV exits as a functional or structural reading frame, concrete function of which needs to be discussed in further research. Innovation of this research is that the ORF3 of DuCV is the first time to be verified as a functional or structural reading frame in the duck by Western Blot.3. Eukaryotic expression of FJ0601 DuCV ORF3The ORF3 gene of duck circovirus (DuCV) including complete open reading frame(ORF) was amplified by PCR, and it was cloned into the pFastBacHT B vector of bac-to-bac baculovirus expression systems to construct the recombinant transfer vector pFast-ORF3. The transfer vector was transformed into DH10Bac competent cell which contain the BmNPV bacmid. A recombinant shuttle vector which contain ORF3 gene was constructed by site-specific transposition and the colonies containing recombinant bacmid were collected by white selection. The cultured sf9 cells were transfected with recombinant bacmid DNA mediating with cellfectin reagent and the pure recombinant baculovirus Bacmid-ORF3 was obtained. After transfecting this transformant into Sf9 cells, ORF3 protein expression in Sf9 cells was detected by western blotting. The experimental results showed that the recombinant buculovirus was obtained and the cap protein with a molecular weight of 17kD was expressed in insect Sf9 cells. The recombinant protein can be recognized by DuCV-positive serum. This would lay a foundation for developing DuCV.