Molecular cloning and analysis of the 3' terminal sequence of duck hepatitis virus genome / 病毒学报

The authentic 3' terminal sequences of genomes of duck hepatitis virus (DHV) serotype I strain C80 and serotype I variant strain E63 were obtained by using 3' RACE and RT-PCR techniques. The analysis showed that 3' terminal sequences in the genomes of the two DHV strains include the 3D region of 1359 nucleotides (nt), the terminator codon TGA, and 3'untranslated region (UTR) of 314 nt. The poly (A) tails of C80 and E63 are 18 nt and 19 nt in length respectively. The deduced 3D proteins of 453 amino acids of both DHV strains contain the motifs KDELR, DxxxxD, GxxCSGxxxTxxxNS, YGDD, and FLKR characteristic for RNA polymerase of picornaviruses, which confirms DHV serotype I to be a member of Picornaviridae . The amino acid sequence identities among 3D protein of the two DHV strains with representatives of nine other picornavirus genera range from 16% to 37%, which are within the value ranges (18%-60%) of 3D amino acid sequence identities obtained from inter-genera comparisons. In addition, DHV serotype I possesses the longest 3'UTR in the family Picornaviridae. Phylogenetic analysis of 3D proteins indicated that DHV serotype I may belong to a separated genus of the family Picornaviridae.

Cloning of CTB-PROIN fusion gene and its expression in Escherichia coli / 生物工程学报

A fusion gene CTB-PROIN, in which Proinsulin gene was fused to the 3' end of CTB gene by a hinge peptide 'GPGP', was constructed and cloned into pET-30a(+) to obtain a prokaryotic expression vector pETCPI. Subsequently the recombinant plasmid pETCPI was transformed into E. coli stain BL21 (DE3). After induced by IPTG, the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE), and its result indicated that the recombinant protein CTB-PROIN was expressed and accumulated as inclusion bodies. The recombinant CTB-PROIN protein accumulated to the level of 25% of total bacterial proteins. After inclusion bodies was denaturalized and refolded in vitro, significant assembly of monomers had occurred, and the recombinant protein represented assembled pentamers. The results of western blotting analysis also demonstrated that the fusion protein could be recognized by the anti-CT and anti-insulin antibody, respectively. In addition, the result of the CTB-PROIN-GM1 binding assay, that the protein could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. These results provided the possibility of developing a cheaper and more efficient oral vaccine for type I diabetes using such constructs.