Cloning, prokaryotic expression and subcellular localization in the infected host cells of the duck plague virus DPV UL35 gene / 病毒学报

Based on the duck plague virus (DPV) UL35 gene sequence that our laboratory obtained (GenBank accession number EF643558), a pair of primers was designed using Oligo6.0 and primer5.0, then the UL35 gene was amplified from DPV CHv strain genomic DNA and cloned into the pMD18-T to construct a clone plasmid pMD18-T-UL35. After identification of the pMD18-T-UL35 by PCR amplification and restriction digestion, the fragment of the UL35 gene was subcloned into the prokaryotic expression vector pET-32a(+). The resultant recombinant plasmid pET-32a(+)-UL35 was then transformed into E. coli BL21 (DE3) strain and optimally-expressed under the induction of 1.0 mmol/L IPTG at 34 degrees C for 5 hours. SDS-PAGE analysis showed the recombinant protein (VP26) had a molecular weight of about 33KDa and accounted for 32.3% of total bacterial protein by gel scanning. The protein was then purified by Ni(2+)-affinity chromatography and used to immunize rabbit for producing the VP26 anti-serum and its antibody titer was up to 1:32 detected by agar diffusion reaction. After the IgG of the polyclonal antibodies was purified by High-Q anion-exchange chromatography, Western blot analysis indicated that the IgG had specific reaction with the VP26. Moreover, the subcellular localization detection was observed using immunofluorescence technique. The results showed that the specific fluorescences appeared relatively few in nucleus in 2 to 8 hours and increased gradually in 12 to 36 hours and eventually reached to the maximum, which aggregated in the spot region of the nucleus after the duck embryo fibroblast (DEF) were infected by DPV. However, there were only a small amount of specific fluorescences in the cytoplasm in 12 hours and increased with the extension of infection time in 24 to 48 hours. The specific fluorescences finally reached to the maximum in the cytoplasm in 72 hours. The results provided significant data for furthering the study on the function of DPV UL35 gene.

Replication of duck plague virus in artificially infected ducks detected by in situ hybridization / 病毒学报

Replication of duck plague virus(DPV) in artificially infected ducks were detected by in situ hybridization (ISH) which employed a 37bp oligonucleotide as probe designed according to DPV DNA sequence in GenBank. The results indicated that DPV DNA was detected in liver, intestine and bursa Fabricius at 4 h, in spleen and esophagus at 6h, in thymus at 12h post infection; DPV DNA in lung and kidney was detected only in dead ducks and no positive signal was detected in muscle, heart, cerebrum and pancreas. DPV DNA was distributed in cell nucleus and cytoplasm. Hepatocytes, sinus endodermal cells and Kuffer's cells were the mainly infected cell types in liver. DPV DNA was mainly detected in epithelium of villi, in lamina propria of intestinal villi of duodenum, in stratum spinosum of esophagus, and in epithelium, cortex, medulla of bursa Fabricius. The positive signals were mainly detected in medulla of thymus, lymphocytes and macrophages of spleen. The research suggests that ISH is a direct and specific method in detecting DPV DNA in paraffin sections and it's also a good method for virus diagnosis and DNA location of DPV.

Prokaryotic expression of pilA gene of type I pilus of enteropathogenic Escherichia coli isolated from ducks and the immunoprotection effect of recombinant expression protein challenged with virulent strains / 生物工程学报

A pair primer was designed by Oligo 6.0 according to the pilA gene sequence of E. coli isolated from human in GenBank. The pilA Gene was obtained by PCR with the enteropathogenic E. coli isolated from ducks as template and cloned into pMD18-T vector. It was identified by PCR, restriction endonuclease analysis, DNA sequencing and then subcloned into BamH I/Hind III site of prokaryotic expression vector pET-32a(+) and recombinant expression plasmid pET-32a-pilA was constructed successfully. The plasmid was transformed into Eschericha coli BL21 (DE3) and 36kD pilA recombinant protein was expressed be induced with IPTG. The protein was purified by Ni-agarose affinity chromatograghy and was prepared as vaccine with Freund' s adjuvant. The ducklings were immunized with the vaccine at 1 and 8-day-old respectively. Two weeks after last immunized, the antibody titer of duck serum was detected by ELISA and the ducklings were challenged with 10(9) PFU enteropathogenic E. coli GH1.2 virulent strain. The immunoprotection effect of pilA recombinant protein vaccine was evaluated according to the mortality, re-isolated rate of E. coli, and grades of pathological changes. The results show that the antibody titer are 1:12800, but 1:200 were detected from ducklings immunized with homologous whole cells E. coli inactivated vaccine. The mortality, re-isolated rate of E. coli, degree of pathological changes of immunized ducklings is lower than that of the control ducklings and showed significant or extremely significant differences (P < 0.01 or P < 0.05), but non-significant difference compared to the ducklings which immunized with homologous whole cells E. coli inactivated vaccine (P > 0.05). The results show that pilA recombinant protein has some immunoprotection effect with the challenging of virulent strains of E. coli GH1.2.