Ⅰ型马立克病毒UL24蛋白的原核表达及其亚细胞定位的研究

Study of Prokaryotic Expression and Subcellular Localization of Serotype Ⅰ MDV UL24 Protein

为研究Ⅰ型马立克病毒（MDV）UL24蛋白的亚细胞定位,以MDV GX0101为模板,PCR扩增UL24基因全长,分别克隆到原核表达载体pGEX-6P-1、pET-32a（＋）和真核表达载体pEGFP-C1中。将原核表达重组质粒转化到大肠杆菌感受态细胞BL21（DE3）中表达,并进行纯化和鉴定。用纯化蛋白免疫Balb/c小鼠,制备并鉴定抗UL24蛋白的多克隆抗体。通过间接免疫荧光试验（IFA）检测感染GX0101的鸡胚成纤维细胞（CEF）中的UL24蛋白。同时,将真核表达重组质粒pEGFP-C1-UL24转染CEF细胞,通过激光共聚焦显微镜观察UL24蛋白在CEF中的亚细胞定位。结果显示Ⅰ型MDV的UL24基因在原核表达载体中能够正确表达,免疫小鼠后获得抗UL24蛋白的多克隆抗体。该抗体可特异性识别MDV UL24蛋白,且MDV UL24蛋白在CEF的细胞质和细胞核中定位,以上研究结果为进一步研究MDV UL24基因的功能奠定基础。

To investigate the subcellular localization of UL24 protein of serotype Ⅰ Marek′s disease virus（MDV）.We use serotype Ⅰ MDV GX0101 as template,PCR amplified UL24 gene and then cloned it into prokaryotic expression vector pGEX-6 P-1,pET-32 a（＋）and eukaryotic expression vector pEGFP-C1 respectively.The recombinant plasmid pGEX-6 P-UL24 and pET-32 a-UL24 was transformed into Escherichia coli BL21（DE3）competent cell respectively,and the expression products were purified and identified.5-week old Balb/c mice were immunized with the purified fusion protein and the anti-UL24 polyclonal antibodies were prepared.The specificity of the polyclonal antibody was identified by indirect immunofluorescence assay（IFA）method.The subcellular localization of UL24 protein in Chicken embryo fibroblast cells（CEF）was observed using a laser scanning confocal microscope.Recombinant eukaryotic expression plasmid pEGFP-C1-UL24 was transfected into CEF cells and the subcellular localization of UL24 protein in CEF cells was observed with a laser scanning confocal microscope.The results showedthat the serotype Ⅰ MDV UL24 gene expressed well with the prokaryotic expression vector.We can get high titer polyclonal antibody that reacted specifically with UL24 protein after immunized mice.The prepared anti-UL24 polyclonal antibody could identify MDV UL24 protein specifically,and showed UL24 protein locates both in cytoplasm and nucleus.This article lays the foundation for further investigation of UL24 protein.