摘要
目的:分离并鉴定石头鱼粗毒液毒性成分,克隆其序列并进行原核表达。方法:利用SDS-PAGE及凝胶过滤HPLC分离石头鱼粗毒液,质谱鉴定其序列;利用RACE技术钓取毒素基因;将获得的毒素cDNA连入pET-22b(+)载体,转化宿主细胞大肠杆菌,经IPTG诱导表达,用亲和层析纯化目的蛋白。结果:从石头鱼粗毒液中分离到NeoVTX等多种蛋白质,克隆了NeoVTXα和β亚基的cDNA序列,获得了纯度为95%以上的重组α亚基蛋白。结论:中国南海石头鱼粗毒液的主要成分为NeoVTX,其α、β亚基序列与日本冲绳海域石头鱼NeoVTX的α和β亚基具有很高的同源性;大肠杆菌菌株可稳定表达α亚基。该工作为NeoVTX抗体制备奠定了基础。
Objective： To isolate and identify protein components from stonefish venom, then clone cDNA sequences coding proteins and express them in prokaryotic cell. Methods： Stonefish venom was isolated by SDS-PAGE and gel filtration HPLC and identified by LC-MS. Gene of toxins was cloned by RACE. cDNA was ligated into pET-22b（＋） vector and transducted to E.coli. Recombinant toxin was induced by IPTG and purified by affinity chromatography. Results： Several proteins including NeoVTX were isolated from stonefish venom, cDNA of α and β subunit of NeoVTX were cloned. Recombinant α subunit was yielded with a purity of more than 95%. Conclusion： Main component of stonefish venom from South China Sea is NeoVTX which shares a high homology with that found in Okinawa, Japan. α subunit could be successfully expressed in E.coli. This work lays the foundation for the preparation of antibody against NeoVTX.
出处
《生物技术通讯》
CAS
2014年第3期345-348,376,共5页
Letters in Biotechnology
基金
国家高技术研究发展计划主题项目(SS2013AA090204)
作者简介
张龙霄（1988-），男，硕士研究生；
刘珠果（1978-），男助理研究员；
通信作者：戴秋云，（E-mail）qy_dai@yahoo．com
