[ Objective] To investigate the immunogenicity of main antigen epitope of RHDV （ Rabbit haemorrhagic disease virus） VP60 expressed in a prokaryotic system. [ Method] The major antigen epitope gene of RHDV VP60 was a...[ Objective] To investigate the immunogenicity of main antigen epitope of RHDV （ Rabbit haemorrhagic disease virus） VP60 expressed in a prokaryotic system. [ Method] The major antigen epitope gene of RHDV VP60 was amplified by RT-PCR. It was cloned into pET-28b （ ＋ ） and expressed in E. coli Rosetta strain. The recombinant protein was detected by Western blot. The pudfied recombinant protein was used to immunize rabbits in order to observe its immunogenicity. [ Result] Western blot analysis revealed a clear band at approximately 24.0 kDa. The purified recom- binant protein reacted with the purified RHDV in ELISA. [ Conclusion] The prokaryotically expressed main antigen epitope of RHDV VP60 shows good immunogenicity.展开更多
Ultrastructural pathological changes in the gut-associated lymphoid tissues of sacculus rotundus (SR) of rabbits infected with rabbit haemorrhagic disease virus (RHDV) were first observed. There were numerous holes at...Ultrastructural pathological changes in the gut-associated lymphoid tissues of sacculus rotundus (SR) of rabbits infected with rabbit haemorrhagic disease virus (RHDV) were first observed. There were numerous holes at the luminal and basement membrane surfaces of the dome epithelium (DE), consistently accompanied by necrosis of lymphocytes and M-cells, and pronounced depletion of lymphocytes in the domes and follicles, decrease of DE complex with formation of pseudomembranous structure on the surface of the dome epithelium. A specific finding in lymphocytes and macrophages was that severe destruction detraction of the membrane of rough endoplasmic reticulum(RER) was accompanied by conspicious increase of solitary, ribo-some-like particles in the cytoplasm, with appearances of intranuclear particles and intranuclear inclusions. It was found that there were many round and dense virion-like particles, with 26 nm in diameter, in the nuclei and cytoplasm of lymphoctes, plasma cells, macrophages and fibroblasts, or in degenerated cells and cellular debris. At the same time, another round virion-like particles about 34 nm in diameter were also seen in the cytoplasm of some cells and interstitium. The results indicated that the appearances of the ribosome-like particles, virion-like particles and inclusion bodies were related to the replication and assembly of RHDV. The present observations suggested that DE of sacculus rotundus could be a open pathway and a transporting route for the entry of antigens into hosts. While the antigen is profoundly deleterious, DE may be as a closed portal or a barrier preventing the foreign antigenic materials from invading.展开更多
为建立鸡新城疫病毒La Sota株在LMH细胞上的全悬浮培养工艺,以获得高滴度的新城疫疫苗抗原,采用LMH贴壁细胞悬浮培养驯化方法,获得了形态良好、能稳定传代的LMH悬浮细胞株;以LMH悬浮细胞在摇瓶培养NDV La Sota株为基础,对接毒细胞密度...为建立鸡新城疫病毒La Sota株在LMH细胞上的全悬浮培养工艺,以获得高滴度的新城疫疫苗抗原,采用LMH贴壁细胞悬浮培养驯化方法,获得了形态良好、能稳定传代的LMH悬浮细胞株;以LMH悬浮细胞在摇瓶培养NDV La Sota株为基础,对接毒细胞密度、接毒剂量、胰酶添加浓度、收获时间等工艺参数进行了摸索和优化,并在14 L生物反应器中进一步进行3个批次的培养验证。结果显示:LMH悬浮细胞以初始密度1.5×10^(6)/mL左右接种,培养72 h可增殖到8.0×10^(6)~9.0×10^(6)/mL,细胞活率达97%以上。确定NDV La Sota株在LMH悬浮细胞株上的培养工艺:LMH细胞悬浮培养至不低于4.5×10^(6)/mL按照感染复数不低于0.2接种病毒,并添加终浓度为5μg/mL的胰酶,于37℃培养72 h左右,细胞活率在30%左右收获病毒液,NDV HA均可达1∶2048~1∶4096,病毒含量≥10^(7.63)EID50/0.1 mL。结果表明成功建立了LMH细胞全悬浮培养工艺,并能在生物反应器扩大培养,为ND相关疫苗研发提供了技术支撑。展开更多
Based on the infectious full-length cDNA clone of rabbit hemorrhagic disease virus (RHDV), the in vitro transcripts are introduced into RK13 cells. 12 h later, CPE could be observed clearly, and virual antigen could a...Based on the infectious full-length cDNA clone of rabbit hemorrhagic disease virus (RHDV), the in vitro transcripts are introduced into RK13 cells. 12 h later, CPE could be observed clearly, and virual antigen could also be detected by IFA. The titre of the recovered virus is 104.6/mL. Immune electron micro-scopic observation of the virus particles revealed that the particles were rotund with a diameter of about 30 nm. Besides, virus titre quantification obtained by qRT-PCR showed a correlation between time from infection and virus titre. All these results showed that we have recovered RHDV from RK13 cells by re-verse genetics technology successfully, and this would be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and new type vaccines of RHDV.展开更多
文摘[ Objective] To investigate the immunogenicity of main antigen epitope of RHDV （ Rabbit haemorrhagic disease virus） VP60 expressed in a prokaryotic system. [ Method] The major antigen epitope gene of RHDV VP60 was amplified by RT-PCR. It was cloned into pET-28b （ ＋ ） and expressed in E. coli Rosetta strain. The recombinant protein was detected by Western blot. The pudfied recombinant protein was used to immunize rabbits in order to observe its immunogenicity. [ Result] Western blot analysis revealed a clear band at approximately 24.0 kDa. The purified recom- binant protein reacted with the purified RHDV in ELISA. [ Conclusion] The prokaryotically expressed main antigen epitope of RHDV VP60 shows good immunogenicity.
基金This work was supported by the National Natural Science Foundation of China(39200092,39870584).
文摘Ultrastructural pathological changes in the gut-associated lymphoid tissues of sacculus rotundus (SR) of rabbits infected with rabbit haemorrhagic disease virus (RHDV) were first observed. There were numerous holes at the luminal and basement membrane surfaces of the dome epithelium (DE), consistently accompanied by necrosis of lymphocytes and M-cells, and pronounced depletion of lymphocytes in the domes and follicles, decrease of DE complex with formation of pseudomembranous structure on the surface of the dome epithelium. A specific finding in lymphocytes and macrophages was that severe destruction detraction of the membrane of rough endoplasmic reticulum(RER) was accompanied by conspicious increase of solitary, ribo-some-like particles in the cytoplasm, with appearances of intranuclear particles and intranuclear inclusions. It was found that there were many round and dense virion-like particles, with 26 nm in diameter, in the nuclei and cytoplasm of lymphoctes, plasma cells, macrophages and fibroblasts, or in degenerated cells and cellular debris. At the same time, another round virion-like particles about 34 nm in diameter were also seen in the cytoplasm of some cells and interstitium. The results indicated that the appearances of the ribosome-like particles, virion-like particles and inclusion bodies were related to the replication and assembly of RHDV. The present observations suggested that DE of sacculus rotundus could be a open pathway and a transporting route for the entry of antigens into hosts. While the antigen is profoundly deleterious, DE may be as a closed portal or a barrier preventing the foreign antigenic materials from invading.
基金This work was supported by the Natural Science Foundation of Zhejiang Province(Grant No.Y305047).
文摘Based on the infectious full-length cDNA clone of rabbit hemorrhagic disease virus (RHDV), the in vitro transcripts are introduced into RK13 cells. 12 h later, CPE could be observed clearly, and virual antigen could also be detected by IFA. The titre of the recovered virus is 104.6/mL. Immune electron micro-scopic observation of the virus particles revealed that the particles were rotund with a diameter of about 30 nm. Besides, virus titre quantification obtained by qRT-PCR showed a correlation between time from infection and virus titre. All these results showed that we have recovered RHDV from RK13 cells by re-verse genetics technology successfully, and this would be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and new type vaccines of RHDV.
