摘要
为获得赤羽病病毒(Akabane virus,AKAV)核衣壳蛋白重组抗原,经RT-PCR扩增了OBE-1株的S节段,将其插入到pMD 18-T载体,然后用带有酶切位点的引物从该重组载体亚克隆出表达核衣壳蛋白的N基因,用BamHⅠ和XhoⅠ双酶切后与同样处理的表达载体pET-28a(+)进行连接,转化感受态细胞BL21(DE3)。将筛选出的阳性克隆进行测序、IPTG诱导表达和融合蛋白纯化及活性鉴定。结果表明,N基因含有1个全长702 bp的ORF,编码233个氨基酸,通过SDS-PAGE和Western-blotting分析,证实重组菌株可高效表达有免疫活性的分子质量约27 ku的可溶性融合蛋白,用薄层扫描仪分析,其最高表达量占可溶性菌体蛋白总量的58.5%。工程菌经超声波裂解高速离心后,上清液在ProBondTMPurification System(含Ni2+)天然状态下纯化,可获得高纯度的融合蛋白。
To obtain a recombinant nucleocapsid （N） protein of Akabane virus for diagnostic purpose, the S fragment from OBE-1 strain was amplified by RT-PCR, The amplified fragment was cloned into a pMD 18-T vector and N gene was subcloned into a pET-28a （＋） expression vector after digestion with BamH Ⅰ and Xho Ⅰ . The recombinant plasmid was transformed into E. coli BL21 （DE3）. The Akabane virus N protein expressed in E. coli induced by IPTG was a fusion protein and was identified by SDS-PAGE and Western-blotting. There was a soluble fusion protein approximately 27 ku with immunological activity. About 58.5% of total soluble protein in host cells was fusion protein and can be purified by ProBondTM Purification System （Ni^2＋） under native conditions.
出处
《中国兽医科技》
CAS
CSCD
北大核心
2005年第12期937-941,共5页
Chinese Journal of Veterinary Science and Technology
基金
中国农业科学院哈尔滨兽医研究所所长基金项目(2005-D1-02)
