机构地区: 广州医学院蛇毒研究所
出 处: 《广州医学院学报》 2006年第1期5-8,共4页
摘 要: 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.
领 域: [生物学]