摘要
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 develop a safe and effective lentlvirus vaccine model and provide insights into the development of other lentivirus vaccines. Methods In this study, a construct of pGPT was made by deleting env gene in the infectious Equine infectious anemia virus（EIAV） molecular clone of WU57. Since the overlaping of EIAV Rev gene with env gene, there was no Rev gene in the construct of pGPT. For compensation of Rev function, the construct of pGPTC was made by inserting 4 copies of constitutive RNA transport elements（CTEs） from Mason-Pfizer monkey virus into the construct of pGPT. In addition, a construct designated pTEB expressing EIAV Env protein was made while env gent-minus viruses were made by co-transfection of pGPT/pTEB or pGPTC/pTEB into 293 cells. Western blot was used to identify the development of recombinant virus particles. Then immunofluorescence assay was used to evaluate the infectivity of recombinant virus particles in vitro. Results EIAV proteins expression was detected in the supernatant of transfected 293 cells by Western blot within pGPTC/pTEB transfeeted cells. However, no evidence of EIAV proteins expression was observed within pGPT/pTEB transfected cells. EIAV proteins expression was detected in the first round but not in the second round infected EK cells with EIAVGPTC by immunofluoreseence assay. Conclusion Rev/RRE was necessary for expression of viral structural proteins; CTEs from Mason-Pfizer monkey virus was functionally interchangeable with EIAV Rev/RRE to help RNAs transportation out of nucleus to express structural proteins and EIAV particles were produced in the transfected 293 cells. A live EIAV recombinant virus with single round infection had been developed.
出处
《中华流行病学杂志》
CAS
CSCD
北大核心
2006年第3期249-252,共4页
Chinese Journal of Epidemiology
