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目的构建单周期复制的马传染性贫血病毒(EIAV)重组疫苗。方法将感染性EIAV分子克隆WU57囊膜(env)基因敲除,在pGPT中克隆4个拷贝的Mason Pfizer猴病毒组成性RNA转运因子(CTE),构建重组质粒pGPTC;构建另一表达Env蛋白的重组质粒pTEB,并分别与重组质粒pGPT、pGPTC共转染进入293细胞;以Western blot对转染细胞外培养液进行检测以鉴定病毒粒子的产生;通过免疫荧光分析鉴定感染细胞内病毒蛋白的表达。结果pGPTC/pTEB共转染的细胞外培养液中检测到特异性EIAV病毒蛋白,而pGPT/pTEB共转染的细胞外培养液中没有检测到特异性EIAV病毒蛋白。转染细胞产生的病毒粒子EIAVGPTC感染的原代马肾细胞(EK)内检测到病毒蛋白的表达,而用感染细胞产生的病毒粒子再感染EK细胞,则无病毒蛋白表达。结论Rev/RRE对于病毒结构蛋白的表达是必需的;CTE可以替代Rev蛋白功能辅助EIAV结构蛋白的表达;转染的293细胞产生了EIAV病毒粒子并分泌到胞外培养液中;产生的病毒粒子EIAVGPTC是一复制缺陷的活重组病毒。
Objective To construct a single-cycle replication-competent equine infectious anemia virus (EIAV) recombinant vaccine. Methods Infectious EIAV molecular cloning was performed to knock out env gene of WU57 gene. Four copies of Mason Pfizer virus constitutive RNA transfer factor (CTE) were cloned into pGPT to construct recombinant plasmid pGPTC. The recombinant plasmid pTEB was co-transfected into 293 cells with the recombinant plasmids pGPT and pGPTC respectively. The expression of the virus particles in the infected cells was identified by immunofluorescence analysis. . Results The specific EIAV virus protein was detected in pGPTC / pTEB cotransfected extracellular culture medium, but no specific EIAV virus protein was detected in pGPT / pTEB cotransfected extracellular culture medium. Virus protein expression was detected in EKVGPTC-infected primary horse kidney cells (EK) from the transfected cells, whereas viral particles from infected cells were reinfected into EK cells without viral protein expression. Conclusions Rev / RRE is necessary for the expression of viral structural proteins; CTE can substitute for the function of Rev protein for the expression of EIAV structural proteins; the transfected 293 cells produce EIAV virions and are secreted into the extracellular culture; the resulting virions EIAVGPTC is a replication defective live recombinant virus.