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

作　　者：张言坤 吴佳燕 韩妮[1,2,3] 周忠文 苏帅 崔治中[1,2,3] 

机构地区：[1]山东农业大学动物医学院,泰安271018 [2]山东省动物生物工程与疾病防治重点实验室,泰安271018 [3]山东省畜禽疫病防制工程技术研究中心,泰安271018

出　　处：《病毒学报》2018年第1期67-74,共8页Chinese Journal of Virology

基　　金：国家自然科学基金河南联合基金(项目号:U1604232),题目:鸡马立克病病毒(MDV)遗传、变异与致病的分子机制;国家自然科学基金青年基金(项目号:31402235),题目:Ⅰ型马立克氏病毒独特基因sorf2生物学功能的研究;山东省"双一流"建设项目(项目号:SYL2017YSTD11)~~

摘　　要：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.

关 键 词：I型马立克病毒(MDV) UL24基因 多克隆抗体 鉴定 亚细胞定位 

分 类 号：S852.65[农业科学—基础兽医学]