One-day-old unvaccinated chicks were inoculated with serotype I virulent strain GA and vaccine strain CVI988, serotype III vaccine strain HVT o f Marek's disease viruses(MDV). By using an indirect immunofluorescence assay of suspensions of peripheral blood mononuclear cells(PBMC) with monoclonal antibo dies(MAb) BA4 and BD8 specific to glycoprotein B of MDV, The dynamic course s of vir emia levels for each of 3 strains from 4 days to 45 days postinfection (PI) were determind. Viremia for virulent strain GA-infected chickens were detected from 4 days PI until days before death of infected chickens, it got its peak at abou t 14 days PI. Viremia in serotype 1 vaccine CVI988-inoculated chickens were mea sured between 4 to 22 days PI, it could be detected from 4 days PI and ended at 18 days PI, it got its peak at 8 days PI. For serotype 3 vaccine HVT-inoculated chickens, viremia could be detected beween 4 to 14 days PI, its peak happened a t about 6 days PI. Such IFA with MAbs BA4 and BD8 could be used to evaluate qu al ity of vaccination process in chicken flocks. The differential dynamic courses o f viremia for different strains may be used to diagnose virulent MDV infection i n vaccinated flocks. The viremia levels measured by IFA with the MAbs and co-cu ltivation assay for plague forming unit(PFU) were compared. The results indicate d that IFA assay of PBMC suspension was more sensitive and specific than co-cu ltivation assay, i.e. viremia levels measured by IFA were 30 to 100-fold higher than that by plague-forming assay in CEF for the same sample.

The envelope gene of ADOL-4817 strain of avian leukosis viru s subgroup J (ALV-J) was amplified by polymerase chain reaction （PCR） and clo ned into TA vector. The sequence analysis results showed that the envelope gene is composed of 1?746 bp, 1?554 bp of which could be translated into 517 amino acids for gp85 and gp37. The molecular weight of envelope protein is 57.7kD. T here are 15 potential glycosylation sites in the envelope protein, 13 of which i s located in gp85. Analysis of sequences of envelope gene indicate that ADOL -4817 showed high degree of sequence identity to other ALV-J strains, and m ost close ly related to the like-envelope gene of endogenous virus EAV-HP but divergent from these of other ALV subgroup A-E . These data support the hypothesis that envelope gene of avian leukosis virus subgroup J maybe acquired by recombination with expressed sequences.

Aim The effect and mechanism of human placental extract(HPE) on the lipoprotein-cholesterol metabolism, peroxidation and the function of platelet aggregation in hyperlipaemia rats were abserved.Methods Wistar rat with hyperlipaemia models were given each HPE 0.4 ml (100 g)-1?d-1 through lavage for 12 days.The serum levels of TG,TC,LDL-C,HDL-C and HDL2-C in its subgroup were measured.The activies of LPO and SOD in both blood and liver tissue were determined .The effect of HPE on lipidosis of liver were abserved by fat dyeing.The levels of 6-keto-PGF1?,TXB2 in plasma and maximum platelet aggregation rate were measured by ELASA. Result The levels of HDL-C and HDL2-C were increased (P

Two Avian leukosis viruses of subgroup J (ALV-J) were isolated from layers by inoculating the sample into chicken embryo fibroblast (CEF) cells and by indirect fluorescent assay (IFA) with ALV-J specific monoclonal antibody JE-9. Sequence comparison indicated the gp85 identities were only in the ranges of 83.4%-87.3% compared with five international reference strains and 86.4%-89.6% compared with eight Chinese strains isolated from white meat-type chickens. The gp37 identities were in the range of 91.8-96.4% compared with the five international strains and 93.4-95.9% compared with the eight domestic strains. When compared with the above thirteen strains, two layers isolates were more close to the prototype HPRS-103 and had less deletion in 3'-Ter than those strains. All strains isolated from white meat-type chickens in China had a deletion in the "E" region of 3'-Ter except these two isolates, suggesting these two ALV-J isolates from layers have different evolution origins from other Chinese isolates from white meat-type chickens.
Animals ; Avian Leukosis Virus ; classification ; isolation & purification ; Base Sequence ; Chick Embryo ; DNA, Viral ; chemistry ; Molecular Sequence Data ; Phylogeny

Objective:To investigate the effect of the musculo-periosteum flap of the sternocleidomastoideus with clavicular periosteum on the reconstruction of extensiveness laryngotracheal defect. Method: Retrospectively studied 49 patients, who admited in our department from 1996 to 2005 years for severe laryngotracheal defect. There were 28 males and 21 females aged from 15 to 69 years old (mean age was 47 years old). The causes of laryngotracheal defect were laryngotracheal stenosis(31 cases) and surgery for thyroid carcinoma invading trachea (18 cases). All of 49 cases were treated with the graft of musculo-periosteum flap of the sternocleidomastoideus with clavicular periosteum and placed a silicon T-tube stenting for 3 to 6 months. Result; All of 49 cases were successfully decannulated with recuperative normal airway patency and effective phonation. The follow up ranged from 2 to 10 years, and the effect of operation was steady. Conclusion:The musculo-periosteum flap of the sternocleidomastoideus with clavicular periosteum is an ideal graft for the reconstruction of cervical extensiveness tracheal defect.

Objective To discuss the frequent etiology of spontaneous sub-cortical hemorrhage and its diag- nosis.Methods The clinical materials of 79 cases of spontaneous sub-cortical hemorrhage were analyzed.Results 56% of the hemorrhage was caused by arterial-venous malformation.48% of the hemorrhage was caused by occult AVM.Conclusion AVM is the most frequent etiology of spontaneous sub-cortical hemorrhage.CTA plays a special role in its diagnosis.

By reverse transcription-polymerase chain reaction (RT-PCR), an open reading frame of pathogenesis-related protein 1 (PR1) was isolated from Panax notoginseng and named as PnPR1. Molecular and bioinformatic analyses of PnPR1 revealed that an open reading frame of 501 bp was predicted to encode a 166-amino acid protein with a deduced molecular mass of 18.1 kD. Homology analysis showed that the deduced amino acid sequence of PR1 protein of Panax notoginseng had a high similarity with other higher plants had the same conservative structure domain of cysteine-rich secretory protein (CAP). The recombinant expressed plasmid pET28a(+)-PnPR1 was expressed in Escherichia coli BL21. The expression conditions were optimized by induction at different times, different temperatures, different IPTG concentrations and different giving times. The optimum expression condition was 0.4 mmol.L-1 IPTG at 28 degrees C for 20 h. The successful expression of PnPR1 provides some basis for protein purification and preparation of the monoclonal antibody.
Amino Acid Sequence ; Cloning, Molecular ; Escherichia coli ; metabolism ; Molecular Weight ; Open Reading Frames ; genetics ; Panax notoginseng ; chemistry ; Phylogeny ; Plant Proteins ; genetics ; metabolism ; Plants, Medicinal ; chemistry ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Alignment

In order to clarify Avian leukosis virus (ALV) characteristics from Chinese native chicken breeds, three ALV JS11C1, JS11C2 and JS11C3 were isolated from Chinese native breed "luhua" by inoculation of DF1 cell culture and detection of p27 antigen. Using PCR amplification of env gene, the amplified gp85 genes were analyzed and compared to all six chicken ALV subgroups reported. The gp85 genes of these three viruses were 1 005bp in length and encoded 335 amino acids, and the gp37 genes were 609bp and encoded 203 amino acids. The homology of gp85 among these three isolated strains was 91.9%-97.0%. Comparing to 18 stains of subgroup A, B, C, D, E published in GenBank, the homology was only in the range of 77.7%-84.6%, significantly lower than the gp85 homology observed within the common chicken subgroups A (88.2%-98.5%), B (91.6%-98.8%), and E (97.9%-99.4%). The gp85 homology compared with subgroup J was only 34.2%-36.5%. These results suggested that three isolated strains from Chinese native breed "luhua" belong to a new subgroup different from all six known subgroups from Chickens, and thus designated as subgroup K.
Animals ; Avian Leukosis ; virology ; Avian Leukosis Virus ; classification ; genetics ; isolation & purification ; metabolism ; Breeding ; Chickens ; genetics ; virology ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; Viral Envelope Proteins ; genetics ; metabolism

In recent years, the prevention and cure of infectious bursal disease (IBD) have become more and more difficult due to the emergence of very virulent strains of infectious bursal disease virus (vvIBDV) and the variant strains of IBDV. In this research, the hybridoma cell lines which secretes anti-idiotypic antibodies against anti-IBDV IgG were established. According to the Jerne's theory of immune network, the use of the anti-idiotypic antibodies as a vaccine will be a new method for the prevention of IBD. In this study, the SPF chickens were inoculated with the IBDV- SD strain, and the bursal was obtained from the died chickens. The bursal was then homogenized and frozen-thawed 3 cycles, and the virus samples were prepared by cane sugar density gradient centrifugation and dialysis. Typical IBDV particles were observed under an electron microscope, and the concentration of the virus protein measured by ultraviolet absorbance spectrophotometry was 10.8 mg/mL. SPF chickens were immunized with the virus and the highly immunized sera were prepared and purified by Sulfuric acid ammonia salt out and Sephadex G-25 chromatography. Then, Balb/C mice of six or eight weeks old were immunized interapertoneally(I. P.) with purified antibodies to IBDV at regular intervals. SP2/0 myeloma cells were fused with the spleencytes from the immunized mice at a ratio of 10:1, in 50% polyethylene glycol (1540) and were then cultured in HAT until all the SP2/0 cells died. The hybridoma cells were selected by ELISA and the highly positive holes were cloned 3 times with the method of limited dilution. Two strains (2B6 strain,5F4 strain) of hybridoma cells were obtained, which were shown by ELISA to steadily secrete anti-IBDV idiotypic antibodies. The chromosome number of the two hybridoma cells were about 88 - 106, 95 in average, and the antibodies secreted belonged to the types of IgG1 and Kappa. Balb/c mice of 3 months old were inoculated I.P. with about 10(7) hybridoma cells per capita, and the ascites were collected 12 days later and the titre of anti-IBDV idiotypic antibodies measured by ELISA was 1 :25600 (for 2B6) and 1:12800 (for 5F4) . The ascites containing the anti-IBDV idiotypic antibodies were emulsified with complete or incomplete Freund's adjuvants, and the anti-IBDV idiotypic antibody vaccine was obtained. SPF and common Jingbai chickens were immunized with the vaccine obtained. The immunized chickens with the vaccine were inoculated with IBDV-SD strain at a dose of 2000 ELD50 after twoimmunizations. All the 10 SPF chickens in the non-immunized group were sick, and 8 of them died; and 5 out of the 50 SPF chickens immunized group got sick and 2 died. All the 10 common Jingbai chickens in the control group were sick, and 6 died; 7 of the 30 immunized common Jingbai chickens got sick and only 1 died. Chi2 analysis showed that the difference between the immunized and the non-immunized groups in both the SPF and the common Jingbai chickens were significant (P < 0.01). Our result indicated that the anti-IBDV idiotypic antibody vaccine well protected chickens and had a great potential in both research and clinical application.
Animals ; Antibodies, Anti-Idiotypic ; biosynthesis ; immunology ; Birnaviridae Infections ; immunology ; Cell Line, Tumor ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Hybridomas ; immunology ; metabolism ; Infectious bursal disease virus ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; Viral Vaccines ; biosynthesis ; immunology

The capsid protein (VP60) gene of RHDV was subcloned into the Pichia expressin vector pPICZ B to express the VP60 protein intracellularly. The recombinant plasmid was initially transformed into a E. coli strain TOP10 F'. After verification of the construct by sequencing, the recombinant plasmid was linearized by Sac I in the 5' AOX1 region and then transformed into Pichia pastoris strain GS115 using the Pichia EasyComp Kit. After selecting and verifing for the insertion of VP60 gene in the genome, two clones of Pichia transformants were select for expression test. The recombinant clones were first inoculate with BMGY in baffled flask at 28-30 degrees C in a shaking incubator (250-300 r/min) until culture reaches an OD600 = 2-6, then resuspend the cell pellet to an OD6oo of 1.0 in BMMY medium to induce expression for 5 days by methanol at a concentration of 0.5% in a 1 liter baffled flask covered with 2 layers of sterile gauze. Collect the cell pellets and break it by acid-washed 0.5 mm glass beads. The expression of recombinant Pichia strains was detected by SDS-PAGE and Western analysis with a polyclonal serum which showed a specific protein band of 60kD. Theses results indicates that the recombinant VP60 produced in Pichia was antigenically similar to the viral polypeptide. Electron microscopic observation of the recombinant Pichia-derived protein revealed the presence of virus-like particles similar in size and appearance to native virus capsids. In the haemagglutination test, the recombinant VLPs, like the native RHDV, also agglutinated human blood type O erythrocytes and could be inhibited by the anti-RHDV polyclonal serum.
Animals ; Capsid ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Hemorrhagic Disease Virus, Rabbit ; genetics ; Pichia ; genetics ; metabolism ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Viral Structural Proteins ; biosynthesis ; genetics ; isolation & purification