【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。更多还原

【Abstract】 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.更多还原