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Objective@#To investigate the best amount of TPCK trypsin in Madin Darby canine kidney (MDCK) cell suspension for the culture of H7N9 avian influenza virus.@*Methods@#Different concentrations of TPCK trypsin were added during the periods of cell growth and virus production. Their effects on cell growth, viability, glucose and lactate metabolism, and hemagglutination titer were monitored every 12 h. Inter-batch differences were analyzed. The amount of trypsin added in the cell growth phase was 0, 1 μg/ml, 2 μg/ml, 4 μg/ml, 6 μg/ml, 8 μg/ml, 10 μg/ml and 15 μg/ml. The amount of trypsin added during the virus production period was 0, 0.5 μg/ml, 1 μg/ml, 1.5 μg/ml, 2 μg/ml and 2.5 μg/ml. When the hemagglutination titers were same, the adding amount was further optimized at different multiplicity of infection (MOI) of 0.001, 0.005, 0.025 and 0.05.@*Results@#No significant linear effects of TPCK trypsin concentration on cell number, viability, and glucose and lactate metabolism were observed. No toxicity to cell growth was observed when TPCK trypsin concentration reached 15 μg/ml. After the inoculation of H7N9 avian influenza virus, the hemagglutination titers in the 1 μg/ml, 1.5 μg/ml, 2 μg/ml and 2.5 μg/ml TPCK trypsin groups reached the peaks at 48 h, which were 1∶26.5. At 60 h, the hemagglutination titers of the latter two groups decreased faster than those of the former two groups. When the MOI was 0.005, the hemagglutination titer of the 1.5 μg/ml group at 48 h was 26.5 higher than 26 in the 1 μg/ml group under the same condition. There were differences between different batches of TPCK trypsin.@*Conclusions@#Adding 1 μg/ml and 1.5 μg/ml of trypsin could better promote the proliferation of H7N9 avian influenza virus, and 1.5 μg/ml of trypsin had a wider range of MOI applicability.
ABSTRACT
Chinese Center for Disease Control and Prevention published three confirmed novel H 7N9 avain influenza virus in-fection cases on March 31 ,2013 .The disease then spread across China and caused extremely high morbility and mortality , which trig-gered extensive attention .These cases were caused by a novel H 7N9 avian influenza virus which was highly mutable .Once the virus a-dapted to human respiratory epithelium receptors , a pandemic outbreak would occur immediately .This article summerizes the research progress on the H7N9 avain influenza, including epidemiologic characteristics , etiology features, clinical manifestation, detection tech-niques as well as theraputic and prevention strategy .
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Objective:To research on protective immunity of omph DNA vaccine against avian Pasteurella multocida in mice.Methods: The omph gene fragment amplified by PCR from avian Pasteurella multocida was cloned into pMD18-T.Subsequently it was subcloned into the eukaryotic expression vector pcDNA3.1(+),and the recombinant plasmid pOMPH was obtained.Then the recombinant plasmid was trans fected into SP2/O cells in vitro.The transcription and expression of target gene were analyzed by RT-PCR,Westem blot analysis and indirect immunofluorescence.Three groups of BALB/c mice(n=16) named pOMPH,pCDNA3.1(+) and PBS were intramuscularly vaccinated with the recombinant plasmid,control vector and PBS respectively.The serum antibodies were detected by indirect ELISA.The spleen lymphocyte proliferation (SLP) and secreted IFN-γof spleen were tested by MTT.The mice were challenged with virulent of avian Pasteurella multocida on week 2 post the third immunization,the protection rate were counted.Results: RT-PCR,Western blot analysis and indirect immunofluorescence showed that the omph gene could be,transfected into SP2/0 cells in vitro and expressed the target protein.Indirect ELISA showed that the levels of antibodies in pOMPH group were most significantly higher than in the other groups(P＜0.01).Spleen lymphocyte proliferation by MTT assay indicated that the SI value induced with avian Pasteurella multocida Omps in pOMPH group was higher than those in pCDNA3.1 (+) and PBS groups (P＜0.05).The IFN-γexperiments(Double-antibodies-sandwich-ELISA)showed that the levels of IFN-γ induced with Omps in the group of pOMPH was mostly higher than in the other control groups apperent(P＜0.01 ).The protection rate of pOMPH(70%) was better than in the other groups.Conclusion: The omph DNA vaccine against avian Pasteurella multocida had been constructed successfully.The DNA vaccine could enhance the immunity level and the protective effect of the vaccinated mice.Present study may be useful for the development of avian Pasteurella multocida vaccine.
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Objective To investigate the role of Ephrin A2 in the regeneration and reinnervation of hair cells in the chick cochlea following kanamycin ototoxicity.Methods 66 newly hatched Roman chickens (3 days old) were randomly divided into experimental group and control groups. Experimental chickens (n=48) received intramuscular kanamycin (200 mg/kg:Sigma,St Louis,MO) for 10 consecutive days and were subsequently sacrificed 2 days before the last injection,and 1,3,7,10,15,21,30,and 60 days after the last injection (n=6 per subgroup). Control chickens (n=18) were untreated and sacrificed 3,13 and 43 days after hatching (n=6 per subgroup). Ephrin A2 protein expression in acoustic ganglia was determined by western blot analysis in all chickens after sacrifice. Results Ephrin A2 protein expression was found and the protein level was almost same in acoustical ganglia of all normal chickens. After kanamycin exposure,the Ephrin A2 protein expression level in the cochlea of the experiment chickens from 2 to 7 days after last kanamycin injection was lower than that in control chickens,respectively. Ephrin A2 expression increased obviously at 15 days after kanamycin last injection. By 30 days after the cessation of kanamycin treatment,the level of Ephrin A2 protein approximated to that in normal control group.Conclusion The expression of Ephrin A2 protein in the acoustical ganglia basically synchronizes with the regeneration and the reinnervation of the hair cells in the chicken cochlea following kanamycin damage,indicating that Ephrin A2 may play an important role in this process.
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Objective:To research on protective immunity of omph DNA vaccine against avian Pasteurella multocida in mice.Methods:The omph gene fragment amplified by PCR from avian Pasteurella multocida was cloned into pMD18-T.Subsequently it was subcloned into the eukaryotic expression vector pcDNA3.1(+),and the recombinant plasmid pOMPH was obtained.Then the recombinant plasmid was transfected into SP2/0 cells in vitro.The transcription and expression of target gene were analyzed by RT-PCR,Western blot analysis and indirect immunofluorescence.Three groups of BALB/c mice(n=16) named pOMPH,pCDNA3.1(+) and PBS were intramuscularly vaccinated with the recombinant plasmid,control vector and PBS respectively.The serum antibodies were detected by indirect ELISA.The spleen lymphocyte proliferation (SLP) and secreted IFN-? of spleen were tested by MTT.The mice were challenged with virulent of avian Pasteurella multocida on week 2 post the third immunization,the protection rate were counted.Results:RT-PCR,Western blot analysis and indirect immunofluorescence showed that the omph gene could be transfected into SP2/0 cells in vitro and expressed the target protein.Indirect ELISA showed that the levels of antibodies in pOMPH group were most significantly higher than in the other groups(P