东亚钳蝎钙通道样毒素的克隆与其在大肠杆菌中的表达

Cloning and Expression of Ca^(2+) Channel Toxin-like Scorpion Venom from Buthus Martensii Karsch in Escherichia coli

目的:克隆东亚钳蝎钙通道样毒素(BmCa)基因,并构建原核表达质粒,在大肠杆菌中重组表达该毒素蛋白。方法:以东亚钳蝎尾毒腺的总RNA为模板,通过RT-PCR技术,扩增并克隆BmCa的cDNA基因,并将该基因重组到PET-GST表达载体中,转化入大肠杆菌BL21并诱导表达,采用N i-SuperChelating Resin柱亲和层析纯化,用小白鼠毒性实验检测融合蛋白的活性。结果:成功克隆BmCa基因并构建原核表达质粒,IPTG诱导后表达分子质量约为33KD的融合蛋白,含量为大肠杆菌总蛋白的25%,经亲和层析得较高纯度的融合蛋白,小白鼠毒性实验表明融合蛋白具有活性。结论:在大肠杆菌中成功表达了有活性的东亚钳蝎钙通道样毒素蛋白,为其功能研究奠定基础。

Objective： To clone and reconstruct a prokaryotic expression plasmid of Ca^2＋ channel toxin-like scorpion venom from Buthns martensii Karsch （BmCa） gene, and express the venom protein in E. coli. Methods： The cDNA of BmCa was obtained by means of RT-PCR from the total RNA of the venom gland. The PCR product was expressed in E. coli BL21 （DE3） using a PET-GST expression system. The fusion protein was purified by Ni-Super Chelating Resin. Acute toxicity assay in mice was used as the method to detect the biological activity of fusion protein. Results： The BmCa gene was cloned and the expression plasmid reconstructed. With IPTG induction, the 33KD fusion protein was efficiently expressed aggregating up to more than 25% of total bacterial protein. After injection of purified fusion protein, the mice showed neurotoxic symptoms. Conclusion： We successfully expressed BmCa in E. coli and obtained biologically active protein.