人、牛和羊叠朊基因的表达及牛叠朊蛋白抗血清的制备  

Expression of doppel(Dpl) gene in human,cattle and sheep and preparation of bovine Dpl antisera

作　　者：刘永生[1] 唐江山[1] 路伟[1] 陈豪泰[1] 孙德惠[1] 才学鹏[1] 张杰[1] 

机构地区：[1]中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室/农业部畜禽病毒学重点开放实验室,兰州730046

出　　处：《中国农业大学学报》2008年第3期14-18,共5页Journal of China Agricultural University

基　　金：国家自然科学基金资助项目(3067156330700597);甘肃省科技攻关重点资助项目(2GS042-A41-001-09);甘肃省自然科学基金资助项目(3ZS051-A25-065);国家博士后科学基金资助项目(20040350585)

摘　　要：叠朊(Dpl)是朊蛋白(PrP)的横向同源物,两者的功能密切相关。本研究旨在制备能够特异检测重组表达的或者动物组织中Dpl的抗血清,以用于研究Dpl的功能及其与PrP的关系。根据已克隆的汉族人、中国黄牛、甘肃土种羊的Dpl基因,将其Dpl成熟蛋白编码区分别定向克隆到表达载体pET-30a(+)上,将重组表达载体转入E.coliBL21(DE3)pLysS,IPTG诱导表达。将表达并纯化的牛重组Dpl免疫4只青紫蓝兔,制备牛Dpl的抗血清,SDS-PAGE分析表明人、牛和羊的重组Dpl在E.coli中获得了高效表达。用Ni-NTA树脂亲和层析和胶洗脱法纯化了上述表达产物,ELISA和West-blot分析表明,制备的牛Dpl抗血清与重组人、牛和羊的Dpl以及牛和羊睾丸组织的Dpl发生了特异性反应,却不与重组人、牛和羊的PrP发生反应。上述结果表明制备的Dpl抗血清可以作为人、牛和羊Dpl的检测试剂和研究材料。The prion-like protein doppel （Dpl） is closely related with the function of prion. An experiment was carried out to prepare the Dpl antisera for specific detection of the recombinant Dpl and the Dpl from animal tissues. The Dpl antisera would be uesed to study DPI function and its relationship with PrP, According to the sequence analysis of cloned Dpl gene in human, cattle and sheep, their mature Dpl encoding region were cloned into pET-30a（ ＋ ） and those recombinant expression vectors were constructed, respectively. The recombinant plasmids were transformed into E, coli BL21（DE3）pLysS and IPTG was used to induce the expression of recombinant Dpl, The four Chinchilla rab- bits were immunized with the purified bovine fused Dpl and antisera was prepared, SDS-PAGE analysis indicated that three different fused protein were overexpressed in E, coll. The fused protein was purified with Ni-NTA resin and gel- elution. ELISA and West-blot analysis indicated the rabbit antisera against bovine Dpl could react specificly with fused Dpl of human, cattle and sheep, and natural Dpl in testicle tissue of cattle and sheep. Dpl antisera could not react with fused PrP of human, cattle and sheep. These results indicated that the cattle Dpl antisera could be used to detect and stud,/on the DOl in human, cattle and sheeo.

关 键 词：传染性海绵状脑病 叠朊 朊蛋白 基因表达 抗血清 

分 类 号：S852.659.7[农业科学—基础兽医学]