Asia 1型口蹄疫病毒前导蛋白(L^(pro))亚细胞定位

Subcellular localization of leader protein(L^(pro)) from foot-and-mouth disease virus serotype Asia 1

为研究Asia 1型口蹄疫病毒(FMDV)前导蛋白(Lpro)亚细胞定位,利用RT-PCR方法获得L基因,将其定向克隆入pEGFP-N1真核表达载体,经PCR扩增、酶切鉴定及序列测定分析,将鉴定为阳性重组表达质粒命名为pEGFP-L。利用脂质体介导法将pEGFP-L转染BHK-21细胞,用荧光显微镜观察和Western blotting方法检测目的基因的表达,经碘化丙啶(PI)染色后在激光共聚焦显微镜下观察Lpro的亚细胞定位。结果表明,成功构建重组表达质粒pEGFP-L;FMDV L基因在BHK-21细胞中得到表达;Western blotting证实表达的Lpro具有反应活性;激光共聚焦显微镜观察发现Lpro在BHK-21细胞中呈弥散性分布。

To research subcellular localization of leader protein（Lpro）from foot-and-disease virus serotype Asia 1,L gene was acquired by RT-PCR and cloned into pEGFP-N1 eukaryotic expression vector directionally.Recombinant expression plasmid was identified by PCR,restriction enzyme digestion analysis,sequencing and named.The correct pEGFP-L was transfected into BHK-21 cells.Fusion protein was observed by fluorescence microscope.Subcellular localization of Lpro was examined with laser confocal microscopy after propidium iodide（PI） staining.Results indicated that Lpro was expressed successfully and could react to Asia 1 FMDV polyclonal antibodies.Laser confocal microscopy showed that Lpro distributed in BHK-21 cells dispersively.This study helped to further understanding the function of Lpro.