中国南海石头鱼毒素NeoVTX的分离纯化、基因克隆及表达

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。

Isolation,Purification, Gene Cloning and Expression of Stonefish Toxin NeoVTX from South China Sea

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.