狂犬病病毒核蛋白基因在大肠杆菌中的高效表达

High-level expression of nucleoprotein gene of rabies virus in Escherichia coli

为获得狂犬病病毒(RV)核蛋白抗原,采用RT-PCR方法从狂犬病病毒ERA株中扩增了RV核蛋白基因,分别亚克隆到原核表达载体pET-28a(+)和pET-32a(+)中,经PCR和双酶切鉴定以及序列测定,重组质粒构建成功。将重组质粒转化到大肠杆菌BL21(DE3)中进行表达,表达的核蛋白再经Ni2+-NTA亲和层析纯化。结果显示,核蛋白在pET-32a(+)中的表达量(30.4%)高于在pET-28a(+)中的表达量(19.4%),培养物中的高纯度核蛋白产量分别为13.6和8.45 mg/L。经Western-blotting检测,不同载体表达的核蛋白均可被兔抗RV多克隆抗体特异识别,表明核蛋白具有良好的反应原性。

To obtain nucleoprotein of rabies virus（RV） for diagnostic antigen, the nucleoprotein gene was amplified from rabies virus ERA strain by RT-PCR and subcloned into vectors pET-28a（＋） and pET- 32a（＋）, respectively. PCR identification, digestion with BarnH Ⅰ ＋ Hind Ⅲ and sequencing analysis showed that the recombinant plasmids were successfully constructed. Then they were transformed into Escherichia coli BL21 （DE3） competent cells for expression. The expressed nucleoprotein was purified using Ni^2＋-NTA Column under denaturing conditions. The expression level of the nucleoprotein gene in pET-32a（＋） （30.4%） was higher than that in pET-28a（＋） （19.4%）. The yield of the highly purified nucleoprotein was about 8.45 mg in pET-28a（＋） and 13.6 mg in pET-32a（＋） per liter bacterial culture, respectively. Result of Western-blotting revealed that the expressed proteins were recognized specifically by rabbit anti-RV polyclonal antibody and had good reactogenicity.