口疮病毒VIR蛋白多克隆抗体的制备及VIR蛋白的亚细胞定位观察

Preparation of polyclonal antibodies against VIR protein of orf virus and observation of subcellular localization of VIR protein

为研究口疮病毒VIR蛋白的生物学特性,构建了含有VIR基因的重组表达质粒pET-VIR和含有绿色荧光蛋白融合真核重组质粒pEGFP-VIR;诱导表达和纯化VIR蛋白,并用纯化的蛋白免疫BALB/c小鼠,制备多克隆抗体,利用间接ELISA测定抗体效价,用Western-blot鉴定抗体的反应原性。利用脂质体介导法将pEGFP-VIR质粒转染绵羊睾丸细胞,4,6-二脒基-2-苯基吲哚(DAPI)进行细胞核染色后激光扫描共聚焦显微镜观察VIR在细胞内的表达和亚细胞定位。结果显示,表达和纯化了含有GST标签的GSTVIR蛋白,主要以可溶性形式存在,其分子质量约27ku,制备的多克隆抗体效价达1∶218;Western-blot分析结果显示,此多克隆抗体有较好的特异性反应;VIR蛋白主要定位于细胞核内。本试验结果为VIR生物学功能的深入研究奠定了基础。

To study the biological characteristics of VIR protein of orf virus,a VIR gene was amplified from orf virus and cloned into pET-28a(+)and pEGFP-N1,respectively.The recombinant plasmids were named as pET-VIR and pEGFP-VIR,respectively.The recombinant VIR protein was expressed well in Escherichia coli by induction of IPTG(1mmol/L),and the expressed VIR protein was purified by affinity chromatography.BALB/c mice were immunized with the purified VIR protein to produce polyclonal antibodies.The titer of prepared polyclonal antibodies was determined by enzyme-linked immunosorbent assay,and the reactionogenicity was detected by Western-blot.Ovine testicular cells were transfected with pEGFP-VIR.The expression of VIR protein in ovine testicular cells was observed with laser scanning confocal fluorescence microscope.The results indicated that the expressed VIR protein with about 27 ku molecular weight mainly existed in a soluble form.The prepared polyclonal antibodies against VIR protein reached a titer of 1∶218,and showed good antibody specificity.Laser scanning confocal fluorescence microscope showed that the VIR protein was mainly localized in the celluar nucleus.The results laid the solid foundation for further studies on biological function of VIR protein.