摘要
利用PCR方法从鸭坦布苏病毒山东分离株(BZ株)扩增整个E基因,全长1 503 bp,克隆到pMD18-T载体中,然后将双酶切目的片段亚克隆入pET-28a(+)载体,构建出重组表达质粒PET28a-E。将PET28a-E转化大肠杆菌BL21(DE3)后,经IPTG诱导可表达出分子量约54.8 kD的蛋白,Western blotting试验呈阳性,表明E蛋白有很好的反应原性。以纯化的表达产物作为包被抗原,鸭坦布苏病毒血清为一抗,HRP标记的羊抗鸭IgG为二抗建立间接ELISA方法。采用该方法对80份送检鸭血清进行检测,并与中和试验进行比较,结果显示,两者的符合率为95.0%,表明该方法具有较好的应用前景。
The whole cDNA of E gene was amplified by PCR from duck Tembusu virus （DTMUV） strain BZ, and cloned into the pMD18-T vector. The fragment was identified by restriction enzymes digestion with EcoR I and Xho I , and then was cloned into the pET-28a （ ＋ ） vector. The recombinant expression plasmid PET28a-E was obtained and transformed into BI21 （DE3）. After the recombinant bacteria were induced by optimal concentration of IPTG, the E fusion protein was proved to get exact expression and have good immunogenicity by SDS-PAGE and Western blotting. Base on the expressed protein an indirect ELISA was established to detect DTMUV antibody in duck serum. The indirect ELISA shared 95.0% coincidence rate with the neutralization test, which showed that this method had a good prospect on the detection of DTMUV antibody.
出处
《浙江农业学报》
CSCD
北大核心
2012年第5期782-786,共5页
Acta Agriculturae Zhejiangensis
基金
山东省优秀中青年科学家科研奖励基金项目(BS2009YY019)
