摘要
根据GenBank中发表的GPMVSF02株的NP基因的核苷酸序列设计合成一对引物,通过RT-PCR扩增JS/1/97/Go株的NP基因,将其克隆入pMD18-T载体,序列测定和分析表明:所扩增的NP基因的核苷酸长为1470bp,共编码490个氨基酸。再将NP基因定向亚克隆至原核表达载体pGEX-6P的多克隆位点,经酶切鉴定后将含有NP基因的阳性亚克隆重组质粒转化到大肠杆菌BL21中,用诱导剂IPTG分别以不同的浓度进行诱导,并在不同的诱导时间进行采样,样品经处理后通过SDS-PAGE、Westernblot和Dot-ELISA进行分析。结果显示:以终浓度为1mmol/LIPTG进行诱导4h表达可达到高峰,表达的融合蛋白大小约为80kD,并具有良好的抗原性。
According to the sequence of NP gene of goose paramyxovirus （GPMV） SF02 isolate strain which published on Genebank, A pair of primers was designed and synthesized, The NP gene of JS/1/97/ Go strain was amplified by means of RT-PCR and cloned into pMD18-T vector, The positive recombinant clone was characterized by enzyme digestion and was sequenced. The sequence analysis showed that NP gene consisted of 1 470 bp nucleotides and encoded 490 amino acids. The NP gene of JS/1/97/Go strain was subcloned into prokaryotic expression vector pGEX-6P, The recombinant plasmid carrying NP gene was characterized by enzyme analysis, The positive recombinant plasmid was then transformed into E. coli. BL21strain, Samples were collected at different induction time and different concentration after induced by IPTG. The specificity of the expressed protein was identified with SDS-PAGE, Western-blot and Dot- ELISA. The result shows： The optimal amount of expressed protein is induced with IPTG at 1 mmol/L concentration for 4 hours. The fusion protein is about 80 kD in size. and has very good antigenicity.
出处
《中国兽医杂志》
CAS
北大核心
2005年第12期3-6,共4页
Chinese Journal of Veterinary Medicine
基金
黑龙江省"十五"攻关课题(GB01B503-02)
关键词
鹅副粘病毒
NP基因
克隆
原核表达
抗原性检测
goose paramyxovirus
NP gene
cloning
prokaryotic expression
detection of antigenicity
