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.

The cell apoptosis induced by duck reovirus in duck embryo fibroblasts / 病毒学报

Cell apoptosis induced by duck reovirus (DRV)in duck embryo fibroblasts (DEF) was ascertained by light microscope and electron microscopy, DNA Ladder, flow cytometry and fluorescent microscopy. Typical morphological apoptotic features including cell shrinkage and condensation, margination of nuclear chromatin were observed under light microscope and the formation of apoptotic bodies by electron microscopy. DNA ladder was shown by DNA fragment analysis at 24-144h post infection. Flow cytometry showed that the cell apoptosis appeared at 24h and reached it's crest-time at 72-96h, decreased at 144h. Fluorescent microscopy showed that the apoptotic cells which showed green fluorescence appeared at 24h, the number of dead cells which showed red fluorescence increased with the time went by. The results above confirmed that the apoptosis of DEF was successfully induced by DRV.

Studies on the propagation characteristics of duck plague virulent virus in duck embryo fibroblasts / 病毒学报

The propagation characteristics of virulent duck plague virus (DPV) in duck embryo fibroblast (DEF) were studied by the method of light microscopy observation of DEF cell culture monolayer, electron microscopy observation of infected DEF cell culture, real-time PCR detecting virus propagation. The results demonstrated that on duck embryo fibroblast a number of plaques were formed by DPV 42 h postinfection. Electron microscopy of the ultrathin section of infected duck embryo fibroblasts demonstrated that the nucleic acid of DPV was round in shape with diameter of 35-45 nm and was often in a cluster in the nucleus of DEF. The nucleocapsid of DPV was round in shape with diameter of 90-100 nm and could be observed both in nucleus and cytoplasm of DEF. The mature DPV which had the structures of envelop and tegument was spherical in shape with diameter of 150-300 nm and was located in cytoplasmic vacuoles. DPV penetrated the DEF cell membrane by direct fusion between the viral envelop and the plasma membrane. Progeny viral nucleic acid was produced in the nucleus and the assembled nucleocapsids obtained the structure of tegument in the cytoplasm and obtained the structure of envelop by budding into the cytoplasmic vesicles. The mature DPV particles were released out of the cell through exocytosis of the cytoplasmic vesicles. Detection of DPV by real-time PCR demonstrated that virus in DEF began its obvious propagation 10 h postinfection and virus amount tended to increase until 30 h postinfection. DPV began to be released into the supernatant 22 h postinfection and the DPV amount peaked 50 h postinfection, when the virus content in DEF and supernatant both underwent approximately 10(3) fold increase. DPV mainly existed in the DEF and the virus content in DEF was 10(2)-10(3) fold than the supernatant.

Studies on porcine reproductive and respiratory syndrome virus morphogenesis in Marc-145 cell and the ultrastructural changes of the infected cells / 病毒学报

Electron microscopy was employed for ultrastructural observation of Marc-145 cells infected with porcine reproductive and respiratory syndrome virus (PRRSV) SC1 strain and studied the virus morphogenesis in infected cells. The results demonstrated that PRRSV was spherical and enveloped. The virion is 45-65 nm in diameter and its nucleocapsid was approximately 25-30 nm. PRRSV entered Marc-145 cells by endocytosis, and replicated in the cytoplasm. The mature viruses were released from infected cells by budding or exocytosis. The main ultrastructural changes of the infected cells were as follows: increased number of cytoplasmic vacuoles, dilated endoplasmic reticulum, mitochondria underwent hyperplasia with its ridges swollen, sloughed, and eventually vacuolated. Typical apoptosis was also observed in the infected Marc-145 cells, which included microvilli sloughing off the cell, appearance of apoptotic bodies and cell fragmentation.

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.