摘要
根据GenBank中登录的猪流行性腹泻病毒(PEDV)经典毒株的S基因序列并参照pMD19-T载体序列,设计了一对分别引入限制性内切酶BamHⅠ和XhoⅠ酶切位点的引物,PCR方法从猪流行性腹泻病毒HB/FN株中扩增得到一条1 080bp的片段。将其连入pMD19-T载体中,经酶切鉴定并测序,测序结果和参考株S基因同源性高达100%。根据抗原性分析选择其抗原性较高的655bp基因片段分别插入到pET-28a(+)、pET-32a(+)、pGEX-6p-1中,构建3种不同的原核表达载体,经合适条件的IPTG诱导后SDS-PAGE分析,结果显示S基因在3种表达载体中均能表达,在pET-28a(+)中表达量最高,表达产物分子质量约为25ku。利用His标签的特性对重组蛋白进行纯化,Western blot证明蛋白可以与PEDV阳性血清产生特异性结合反应。用纯化的蛋白免疫小鼠,抗体检测显示S蛋白具有良好的免疫原性。
According to the S gene sequence of porcine epidemic diarrhea virus classic strain in GenBank and PMI）-19T vector map, a pairs of primers were designed which were inserted into the restriction endonuclease BarnH I and Xho I sites. Using PCR technology, a 1 080 bp fragment in suspected porcine epidemic diarrhea disease HB/FN strain was obtained and connected into PMD19-T vectors. By enzyme digestion and DNA sequencing, the results showed that its homology with S gene in GenBank was up to 100%. Based on the antigenicity analysis, 655 bp fragment of higher antigenicity was inserted into pET-28a （＋）, pET-32a（＋） and pGEX-6p-1 to construct prokaryotic expression vector, and the first has the highest ex- pression level. Induced by IPTG, SDS-PAGE analysis showed that S gene could be expressed by these vectors, the product is about 25 ku, the same with expected results. After purification, Western-blot assay indicated that the target protein could react with PEDV immune serum. Antibodies of the mice immunized with purified proteins showed good antigencity.
出处
《动物医学进展》
CSCD
北大核心
2013年第5期21-25,共5页
Progress In Veterinary Medicine
基金
国家自然科学基金项目(31272593)
