鸭坦布苏病毒E基因的克隆表达及初步应用

摘要

The whole cDNA of ￡ gene was amplified by PCR from duck Tembusu virus (DTMUY) 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 BL21 ( 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.%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.