摘要
将水貂阿留申病毒ADV-G株VP2基因中主要抗原表位区的2个片段,分别克隆至原核表达载体pET-28a(+)的多克隆位点中,鉴定后得到重组质粒pET-VP2a和pET-VP2b,将重组质粒转化到宿主菌BL21中,用IPTG分别以不同浓度,不同诱导时间进行诱导表达,采集样品做SDS-PAGE、Western-blot分析,并以此蛋白作为包被抗原进行ELISA检测。结果发现以终浓度为1mmol/L的IPTG进行诱导,4h后表达可达到高峰,其大小分别约为23kD和28kD,该蛋白与ADV阳性血清能发生特异性反应。将此表达产物应用Ni-NTAHis-Bind纯化系统进行纯化,其纯度可达到91%,纯化后的蛋白以60μg/孔包被96孔板,检测不同地区水貂血清,同时以对流免疫电泳法(CIEP)为对照,结果发现二者的符合率高达93%,而应用该蛋白进行检测其反应更为安全,背景低,制备简单,这为今后应用该蛋白作诊断抗原检测水貂阿留申病奠定了基础。
The recombinant expression vectors pET-VP2a and pET-VP2b were constructed by cloning VP2 gene of Aleutian mink disease parvovirus （ADV-G） into a prokaryotic expression plasmid pET-28a. The recombinant vectors were transformed into recipient germs BL21. Samples were collected at different induction time after induction with IPTG. The specificity of the expressed proteins were identified with SDS-PAGE, Western Blotting . And the protein were about 23kDa and 28 KDa in size. The expressed protein were purified byNi-NTA His-Bind purification system and the degree of purity is about 91%.The purified protein was coated on 96-well plate at 60μg/well and used for identification of ADV-positive sera from different regions. The results are 93% identical to CIEP. But this method is safer and easier to perform. High efficient expression of ADV protein will benefit for diagnosis of ADV in practice.
出处
《中国农学通报》
CSCD
北大核心
2010年第8期7-11,共5页
Chinese Agricultural Science Bulletin
基金
科技部科研院所社会公益研究专项"野生动物重要传染病生态学发生
监测和控制技术"(2005DIB4J048)
关键词
水貂阿留申病毒
VP2基因
原核表达
Aleutian mink disease parvovirus
VP2 genes
Prokaryotic expression
