稳定表达小反刍兽疫病毒N蛋白的CHO细胞系的建立及表达产物的抗原性鉴定  

The establishment of CHO cell lines stable expressing Peste des petits ruminants virus N protein and the identification antigenicity of the product expressed

作　　者：李翠翠[1] 孙一瑞[1] 王子龙[1] 陈伟业[1] 步志高[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2016年第4期262-265,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家国际合作专项(2015DFA31300);公益性行业(农业)(201203056)

摘　　要：为制备具有天然抗原活性的小反刍兽疫病毒(PPRV)核衣壳(N)蛋白,本研究建立了稳定表达PPRV N蛋白的真核细胞系。首先,将去除核定位信号(NLS)编码序列并且在其5'端引入组织纤维蛋白溶酶原抗原信号肽(tPA SP)编码序列的PPRV N基因克隆至真核表达质粒pCAGG-neo中,构建了重组质粒pCAGG-N△NLS/his。并将其转染于CHO细胞中,经G418压力培养并采用间接免疫荧光试验(IFA)及western blot试验筛选鉴定,获得稳定表达PPRV N蛋白的细胞系。通过IFA及western blot试验鉴定His标签表明该细胞系传代至22代仍能够稳定表达PPRV N蛋白。最后通过western blot等试验表明该蛋白能够与绵羊抗PPRV多克隆抗体反应,进一步表明该细胞系表达的PPRV N蛋白具有天然的N蛋白抗原性,为PPRV病原学诊断等相关研究奠定了基础。In order to preparation Peste des petits ruminants virus（PPRV） nucleocapsid protein（N） with natural immunogenicity, the cell lines with stable expressing PPRV N protein was established in this study. Firstly, the sequence encoding signal peptide of the tissue plasminogen antigen was introduced to the 5＇ end of PPRV N gene with nuclear localization signal deletion,which was cloned into the eukaryotic expression plasmid of p CAGG-neo to construct the recombinant plasmid of p CAGG-N△NLS/his.Then, p CAGG-N△NLS/hiswas transfected into CHO cells, and the cells expressing PPRV N protein were selected in present of G418,which stably expressed the N protein at least for 22 passages. Identification of the N protein expressed cell line shown that the N protein was able to be recognized by polyclonal antibody against PPRV through western blot and indirect immunofluorescence assay. The established cell line laid the foundation for diagnosis research on PPRV.

关 键 词：小反刍兽疫 核衣壳蛋白 CHO细胞 细胞系 

分 类 号：S852.65[农业科学—基础兽医学]