CELO病毒fiber1基因部分片段的原核表达及抗原性鉴定  

Cloning and expression of protein fiber1 gene of chicken embryo lethal orphan virus and identification of expressed protein antigenicity

作　　者：王凤雪[1] 王云峰[1] 智海东[1] 王玫[1] 童光志[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：《中国兽医科技》2005年第6期468-471,共4页Chinese Journal of Veterinary Science and Technology

基　　金：国家高技术研究发展计划(863)项目(2003AA213020)

摘　　要：根据鸡胚致死孤儿(CELO)病毒Phelps株的fiber1基因(GenBank序列号U46933)序列设计了1对引物,采用PCR扩增CELO病毒fiber1基因C端(包含Knobs区)的部分片段,并将其克隆到质粒表达载体pGEX6p1,获得的阳性克隆命名为pGEXAF807。序列分析表明,所获得的DNA片段核苷酸序列与CELO病毒Phelps株的fiber1基因序列完全一致。将pGEXAF807转化大肠埃希氏菌DH5α,经37℃、0.6mmol/LIPTG诱导表达5h,SDSPAGE分析超声裂解后的上清和沉淀。结果显示,目的蛋白以包涵体形式存在,蛋白分子质量约为54.6ku,目的蛋白量占菌体总蛋白的35.7%。用CELO病毒多克隆血清做Westernblotting试验,结果表明目的蛋白具有较好的抗原活性。With a pair of primer designed accordingto fiber1 gene sequence of chicken embryo lethal orphan virus(CELOV) Phelps strainin GenBank (U46933), a fragment of 807bp was amplified by PCR,and cloned into pGEX-6p-1 vector. The sequence analysis revealed that the target gene has the same sequence as fiber1 gene of CELOV reportedin GenBank.The recombined plasmid was transformed into Escherichia coli DH5αand induced by 0.6~mmol/L of IPTG for 5h. The SDS-PAGE analysis showed that the expressed fusion protein with a molecular weight of 54.6ku existed in the include body. The fusion protein is of 35.7% in total bacterial protein.The Western-blotting analysis showed that the protein has good antigenicity.

关 键 词：鸡胚致死孤儿病毒 fiberl基因 原核表达 抗原活性 诊断试剂盒 包涵体肝炎 

分 类 号：S852.659.1[农业科学—基础兽医学] S858.31[农业科学—兽医学]