表达EGFP报告基因口蹄疫病毒亚基因组复制子的构建

Construction of sub-genomic replicon of foot-and-mouth disease containing EGFP reporter gene

【目的】构建含有EGFP报告基因的口蹄疫病毒(FMDV)亚基因组复制子系统。【方法】利用融合PCR方法,将EGFP报告基因替换O型FMDV全长c DNA克隆中的前导蛋白Lb和结构蛋白P1基因,构建含有EGFP报告基因的FMDV亚基因组复制子FMDV-EGFP。复制子质粒连续转化、测序检验复制子载体的稳定性。Not I线性化的复制子FMDV-EGFP用脂质体介导法转染表达T7 RNA聚合酶的BSR/T7细胞后,不同时间段观察EGFP荧光表达情况。转染的细胞用流式、间接免疫荧光、RT-PCR和Western blot检测该复制子载体的自主复制能力和口蹄疫病毒蛋白的表达情况。【结果】复制子质粒的连续转化及测序表明报告基因可以稳定存在。FMDV-EGFP复制子转染BSR/T7细胞3 h后在荧光显微镜下能够看到绿色荧光,EGFP荧光信号随着转染时间的延长逐渐增加,并且荧光信号可持续6 d以上。转染24 h后的细胞流式分析显示转染的细胞中有6.0%发出荧光,说明构建的复制子载体能够有效表达EGFP蛋白。另外,间接免疫荧光、RT-PCR和Western blot方法也检测到该复制子RNA在BSR/T7细胞中能够进行自主复制,并且能够表达病毒的非结构蛋白。【结论】含有EGFP报告基因的FMDV亚基因组复制子的成功构建为进一步研究病毒复制、翻译机制及筛选抗病毒药物等奠定了坚实的基础。

[Objective] To construct sub-genomic replicon system of foot-and-mouth disease containing EGFP reporter gene. [Methods] Based on the infectious clone of serotype O FMDV, the subgenomic replicon FMDV-EGFP was constructed by replacing Lb and P1 gene of FMDV with EGFP reporter gene using fusion PCR method. The stability of replicon was detected by a series of transformation and sequencing. The replicon linearized with Not I was transfected into BSR/T7 cells expressing T7 RNA polymerase using liposome mediation. EGFP expression of different time of transfected cells was examined using fluorescence microscopy, the ability of self-replication and expression of protein of replicon were detected by flow analysis, immunofluorescence assay, RT-PCR and Western blot. [Results] The replicon vector was stable by a serious of transformation and sequencing. After 3 h transfection, the EGFP fluorescence could be obviously observed under the fluorescence microscope, the level of EGFP protein was increased gradually as transfection time went on. The result of flow cytometry analysis showed that 6.0% transfected cells were able to generate fluorescence, indicating the effective expression of EGFP protein of replicon vector. In addition, the results of immunofluorescence, RT-PCR and Western blot assay demonstrated that the replicon could autonomously replicate and express non-structural protein of FMDV. [Conclusion] The construction of sub-genomic replicon of FMDV expressing EGFP reporter gene provides a foundation to study viral replication and translation mechanism and antiviral drugs.