猪血凝性脑脊髓炎病毒PK细胞膜受体的鉴定  

Identification of Hemagglutinating encephalomyelitis virus Receptor in PK Cells Membrane

作　　者：陆慧君[1] 贺文琦[1] 宋德光[1] 刘立国[1] 常灵竹[1] 李志萍[1] 陈克研[1] 高丰[1] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062

出　　处：《农业生物技术学报》2008年第1期20-23,共4页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.30671551);吉林省科技发展计划重点项目(No.20060206-2)

摘　　要：在毕赤酵母(Pichia pastoris)中表达猪血凝性脑脊髓炎病毒(Hemagglutinating encephalomyelitis virus,HEV)67N株S蛋白片段,纯化后免疫家兔,获得兔抗HEV-S蛋白特异性抗体;提取PK细胞膜蛋白,SDS-PAGE电泳后,转印NC膜,以纯化的S1蛋白代替病毒,利用改进的病毒铺覆蛋白结合试验(VOPBA)对PK细胞膜受体进行鉴定,结果扩增获得1797bp的S1目的基因片段,用S1特异性引物和AOX1通用引物PCR鉴定结果表明,实验成功地构建了重组酵母表达质粒,重组酵母菌经1%甲醇诱导,在培养基上清中检测到73kD目的蛋白。Western blot分析发现,该重组蛋白可与HEV多克隆抗体发生特异性血清反应;提取的PK细胞膜蛋白转膜后与纯化的S1目的蛋白结合,DAB显色发现在90kD处有清晰的蛋白带。To identify Hemagglutinating encephalomyelitis virus （HEV） 67N receptor in PK cell membrane, polyclonal antibody to HEV was prepared with immunizing rabbits by injecting the purified S1 protein; after SDS-polyacrylamid gel electrophoresis （SDS-PAGE）, the PK cell membrane proteins were transferred to nitrocellulose membrane; and virus overlay protein binding assay （VOPBA） was performed by the recombinant S1 protein to identify the receptor protein binding HEV-S1. The results showed that the 1 797 bp S1 gene fragment was obtained by PCR amplification. Identification results by PCR with S1 special primers and AOXI primers suggested that the recombined yeast expression plasmid was constructed successfully. After the recombined strains were induced by 1% methanol, a molecular weight of 73 kD in medium supernatant could be specifically recognized by polyclonic antibody against HEV. After transmembrane, the PK cell membrane proteins were bound with the purified S1 protein, and one clear protein band of 90 kD was found by DAB colouration.

关 键 词：血凝性脑脊髓炎病毒(HEV) 病毒铺覆蛋白结合试验(VOPBA) 受体 

分 类 号：S188[农业科学—农业基础科学]