犬瘟热病毒磷蛋白的表达及抗体制备  

Expression and Antibody Preparement of Canine distemper virus Phospho-protein(P)

作　　者：王晓杜[1] 袁庄川 梅匀安 张敏[1] 周圻[1] 

机构地区：[1]浙江农林大学动物科技学院,临安311300

出　　处：《农业生物技术学报》2014年第4期495-501,共7页Journal of Agricultural Biotechnology

基　　金：浙江省自然科学基金(No.Y3110124);浙江省"十二五"重点学科动物营养与饲料科学

摘　　要：犬瘟热病毒(Canine distemper virus,CDV)是一种急性高度接触性传染病病原,可引起犬(Canis)、猫(Felis)等多种属动物发热、腹泻、肺炎和中枢神经系统紊乱,其磷蛋白(phospho-protein,P)在病毒复制和致病中具有重要作用。本研究根据犬瘟热病毒P蛋白基因(GenBank No.AF164967)设计引物,RT-PCR扩增得到CDV-P基因片段,亚克隆到原核表达载体pET-28(a)上,构建重组原核表达质粒pET-28(a)-CDV-P,转化大肠杆菌(Escherichia coli)BL21(DE3)菌株,异丙基硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)诱导表达重组his-CDV-P蛋白,His-band纯化后获得高纯度重组蛋白,纯化蛋白免疫balb/c小鼠,制备了小鼠抗CDV-P抗体,检测CDV感染细胞内P蛋白的表达和细胞定位,检测不同感染时间细胞内P蛋白的表达。SDS-PAGE电泳结果表明,获得重组蛋白的分子量大小为65 kD,表达的蛋白以包涵体形式存在,占总菌体蛋白的35%以上,纯化后占总蛋白60%以上;Western blot检测表明,小鼠抗CDV-P抗体能特异性识别原核表达产物和病毒感染表达产物,CDV-P在感染细胞内具有两种表现形式(磷酸化和非磷酸化);间接免疫荧光结果显示,该蛋白在胞浆和胞核中都存在;CDV-P在病毒感染的早期和晚期都有表达,随着感染时间延长表达逐渐增加,并且其磷酸化水平也逐渐增加。本研究制备了重组P蛋白和多克隆抗体,检测了P蛋白在感染细胞内的表达和定位,为P蛋白功能和CDV致病机理研究提供基础资料。Canine distemper virus （CDV） is a pathogen of acute infectious disease, which caused fever, diarrhea, pneumonia and central nervous system disorders in Canis and Felis. The phosphorylation protein （P） is important to replication and pathogenicity of CDV. In this study, according to CDV-P gene （GenBank No. AF164967）, primers were designed by primer primer 6.0 software. The P gene was cloned by reverse transcriptional polymerase chain reaction（RT-PCR）, and subcloned into plasmid of pET-28（a）. The pET-28（a）-CDV-P was constructed and identified by enzyme digestion and sequencing. The pET-28（a）-CDV-P was transfected into Escherichia coli BL21 （DE3）. The his-CDV-P was expressed in E.coli BL21 （DE3） by isopropyl-β-D-thiogalactopyranoside （IPTG） inducing. High purified protein was obtained by his-band Ni＋ affinity chromatography. The mouse anti-CDV-P antibodies were prepared from balb/c mouse immunized by his-CDV-P protein . In CDV infected cells, the expression of CDV-P protein was detected by Western blot, and subcellular localization was detected by indirect immunofluorescence assay using mouse anti-CDV-P antibodies. The results showed that his-CDV-P protein was expressed by inclusion body and its molecule weight was 65 kD by SDS-PAGE. The expression of his-CDV-P protein was more than 35% of total bacterial protein, and up to 60% of total protein after purification by grayscale scanning analysis. Mouse anti-CDV-P antibody specifically recognized the prokaryotic expression products and viral infections products by Western blot. In the CDV infected cells, the P protein expressed in the cytoplasm and nuclei by indirect immunofluorescence assay. The results showed that the P potein had 2 forms （phosphorylation and non-phosphorylation） and CDV-P protein expressed in the beginning phase and late phase of post-infection, and the phosphorylation and expression level of CDV-P were gradually increasing in infection stage by Western-blot. In this study, recombinant protein CDV-P and

关 键 词：犬瘟热病毒 磷蛋白 原核表达 病毒感染 细胞定位 

分 类 号：S852.655[农业科学—基础兽医学]