• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

新型鸭呼肠孤病毒σB蛋白的可溶性原核表达与纯化

Prokaryotic Expression and Purification of σB of Novel Duck Reovirus

  • 摘要:
    目的 以新型鸭呼肠孤病毒(novel duck reovirus, NDRV)NP03株σB基因为研究对象,通过原核表达体系优化,旨在获得可溶性表达的NDRV σB蛋白,为该病毒的检测及防治提供参考。
    方法 将双酶切鉴定后的重组质粒pET-32a-σB转化大肠杆菌BL21(DE3)感受态细胞中,使用IPTG诱导表达重组σB(rσB)蛋白。菌体超声破碎后,SDS-PAGE分析rσB蛋白的表达情况。进一步利用His标签蛋白纯化试剂盒对上清液中的rσB蛋白进行亲和层析纯化,并以纯化后的rσB蛋白作为包被抗原建立间接ELISA检测方法。
    结果 在0.5 mmol·L−1 IPTG、16 ℃条件下诱导12 h时,超声破碎后的上清液中可检测到目标蛋白,rσB蛋白的可溶性表达水平显著提高。SDS-PAGE分析显示纯化的rσB蛋白纯度高于90%,分子量约为60 kDa。Western blot结果显示,纯化的rσB蛋白可与His标签单克隆抗体特异性反应;基于该蛋白建立的间接ELISA检测方法显示,NDRV阳性血清的OD450nm值为1.75,显著高于番鸭呼肠孤病毒和番鸭小鹅瘟病毒等对照病毒阳性血清(OD450nm均小于0.3)。Western blot和ELISA结果表明,纯化后的rσB蛋白具有较好的特异性的反应原性。
    结论 通过低温与低浓度IPTG诱导,成功建立了NDRV σB蛋白的可溶性原核表达体系,突破了该蛋白因包涵体形式表达导致的功能与应用研究瓶颈,为开发NDRV血清抗体ELISA检测试剂盒奠定了物质基础。

     

    Abstract:
    Objective The soluble σB of novel duck reovirus (NDRV) was obtained with optimized prokaryotic expression for effective disease detection and control.
    Method The recombinant plasmid pET-32a-σB identified by double digestion was transformed into Escherichia coli BL21(DE3) competent cells. IPTG was used to induce the expression of recombinant σB (rσB). After ultrasonic crushing, the supernatant and precipitate were analyzed by SDS-PAGE. The rσB in the supernatant was then purified by a His-tag protein purification kit to establish an indirect ELISA for the viral detection.
    Result Under the conditions of 0.5mmol·L−1 IPTG and induction at 16℃ for 12h, the rσB was detected in the supernatant with significantly improved expression. The SDS-PAGE analysis showed the purity of rσB to be higher than 90%, and the molecular weight approximately 60 kDa. The purified protein specifically reacted with His-tag monoclonal antibody as shown by western blot. The OD450nm of the NDRV positive serum tested by the indirect ELISA was 1.75, which was much higher than that of positive sera from Muscovy duck reovirus and Muscovy duck goose parvovirus. The OD450nm of MDRV and MDGPV positive sera were both less than 0.3. A strong specific reactivity of the purified rσB was confirmed by both western blot and ELISA.
    Conclusion The optimized prokaryotic expression conditions of NDRV σB protein established in this study applied low temperature and IPTG concentration to successfully induce the soluble NDRV σB protein that overcame the previously existing bottleneck for the functional and application studies. The secured soluble rσB would lead to the future development of NDRV serum antibody ELISA kit.

     

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