Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF) is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV) also does this,but the mecha...Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF) is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV) also does this,but the mechanisms and particular receptors remain unclear.Virus overlay protein blot assays were carried out on CEF membrane proteins,which were extracted respectively with a Mem-PER TM kit,a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method,and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells,indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.展开更多
[Objective] To observe whether fowlpox virus （FPV） can proliferate in chicken embryo passage fibroblasts or not and then try to use chicken embryo passage fibroblasts to replace primary chicken embryo cells for FPV ...[Objective] To observe whether fowlpox virus （FPV） can proliferate in chicken embryo passage fibroblasts or not and then try to use chicken embryo passage fibroblasts to replace primary chicken embryo cells for FPV culture. [Method] Primary chicken embryo fibroblasts were prepared and subcultured. After FPV were inoculated on the 20th passage fibroblasts, cytopathy was observed. Then, the FPV culture was identified and determined quantificationally. [Result] Specific cytopathy appeared in the FPV-inoculated chicken embryo passage fibroblasts. The titer of the yielded FPV culture reached the standard for production of fowl pox vaccine. Further analysis reveals that the chorioallantoic membrane lesions were caused by FPV. [ Conclusion] FPV can reproduce in chicken embryo passage fibroblasts, and the Uter of FPV cell culture can meet the pro- duction requirements of fowl pox vaccine.展开更多
鸡胚成纤维细胞(CEF)是多种动物及人类病毒的易感细胞;也是研究鸡的基因组功能的理想材料.本试验的目的是研究非复制型腺病毒载体对CEF的转染效率及安全性;并建立在CEF细胞中进行RNAi的技术平台.采用GFP重组非复制型腺病毒载体(Ade-GFP...鸡胚成纤维细胞(CEF)是多种动物及人类病毒的易感细胞;也是研究鸡的基因组功能的理想材料.本试验的目的是研究非复制型腺病毒载体对CEF的转染效率及安全性;并建立在CEF细胞中进行RNAi的技术平台.采用GFP重组非复制型腺病毒载体(Ade-GFP)转染CEF细胞;结果证明0.1-2000 MOI Adv-GFP均可转染CEF细胞并表达GFP;转染后16h开始出现GFP阳性细胞;胞核与胞浆可见明亮的荧光;荧光细胞数量及荧光强度在24～36h达到高峰;以后逐渐衰减;约180h全部消失;转染效率最高为22.3%;形态学观察及流式细胞仪测定结果显示;Adv-GFP转染的CEF细胞未见细胞病变;多数转染组细胞活力不受影响;说明用Adv-GFP携带外源转染CEF细胞是安全可行的;用脂质体转染试剂转染化学合成的GFP-siRNA;成功地干涉了腺病毒载体介导的GFP基因在鸡胚成纤维细胞的表达;干涉效率为85%;证明了鸡胚成纤维细胞中存在RNAi机制;为进一步利用非复制型腺病毒载体递送siRNA在CEF细胞内进行RNAi的研究奠定了基础.展开更多
基金supported by a grant from Yunnan Provincial Education Board(08C0070)a grant from Yunnan Provincial Program for Introducing High-level Scientists (2009CI125)
文摘Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF) is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV) also does this,but the mechanisms and particular receptors remain unclear.Virus overlay protein blot assays were carried out on CEF membrane proteins,which were extracted respectively with a Mem-PER TM kit,a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method,and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells,indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.
基金Liaoning Agricultural College for providing test site and fund for Doctors of Liaoning Medical College
文摘[Objective] To observe whether fowlpox virus （FPV） can proliferate in chicken embryo passage fibroblasts or not and then try to use chicken embryo passage fibroblasts to replace primary chicken embryo cells for FPV culture. [Method] Primary chicken embryo fibroblasts were prepared and subcultured. After FPV were inoculated on the 20th passage fibroblasts, cytopathy was observed. Then, the FPV culture was identified and determined quantificationally. [Result] Specific cytopathy appeared in the FPV-inoculated chicken embryo passage fibroblasts. The titer of the yielded FPV culture reached the standard for production of fowl pox vaccine. Further analysis reveals that the chorioallantoic membrane lesions were caused by FPV. [ Conclusion] FPV can reproduce in chicken embryo passage fibroblasts, and the Uter of FPV cell culture can meet the pro- duction requirements of fowl pox vaccine.
文摘鸡胚成纤维细胞(CEF)是多种动物及人类病毒的易感细胞;也是研究鸡的基因组功能的理想材料.本试验的目的是研究非复制型腺病毒载体对CEF的转染效率及安全性;并建立在CEF细胞中进行RNAi的技术平台.采用GFP重组非复制型腺病毒载体(Ade-GFP)转染CEF细胞;结果证明0.1-2000 MOI Adv-GFP均可转染CEF细胞并表达GFP;转染后16h开始出现GFP阳性细胞;胞核与胞浆可见明亮的荧光;荧光细胞数量及荧光强度在24～36h达到高峰;以后逐渐衰减;约180h全部消失;转染效率最高为22.3%;形态学观察及流式细胞仪测定结果显示;Adv-GFP转染的CEF细胞未见细胞病变;多数转染组细胞活力不受影响;说明用Adv-GFP携带外源转染CEF细胞是安全可行的;用脂质体转染试剂转染化学合成的GFP-siRNA;成功地干涉了腺病毒载体介导的GFP基因在鸡胚成纤维细胞的表达;干涉效率为85%;证明了鸡胚成纤维细胞中存在RNAi机制;为进一步利用非复制型腺病毒载体递送siRNA在CEF细胞内进行RNAi的研究奠定了基础.
