Imani Mehdi, Jaliani Hossein Zarei, Kheirandish Mohammad Hassan, Azadpour Mahnaz
Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran.
Iran J Basic Med Sci. 2017 Apr;20(4):380-385. doi: 10.22038/IJBMS.2017.8577.
A newly-introduced protein toxin from a sea anemone, namely fragaceatoxin C is a protein with molecular weight of 20 kDa and pore-forming capability against cell membranes has recently grasped great attentions for its function. In this study, its coding sequence cloned as a fusion protein with His-tag for simple production and rapid purification.
After PCR amplification using and -harboring primers, the gene fragment was cloned into . was used for expression of constructed vector and toxin expression was verified by SDS-PAGE. For one-step purification Ni-NTA sepharose affinity chromatography was employed. For examination of purified toxin function, RBC hemolytic test was conducted.
The results showed that the FraC-coding gene was successfully cloned between and restriction sites and purified with affinity chromatography. Densitometric analysis represented the purity of approximately 97%. Hemolytic test indicated the purified FraC had remarkable lytic activity on RBC and almost lysed 50% of cells at the concentration value of 6.25 nM.
The results indicated that not only purified toxin preserved its activity during expression and purification processes but also exerted its function at lower concentrations so that even the 0.09 nM displayed hemolytic effect.
一种新引入的来自海葵的蛋白质毒素，即脆海葵毒素C，是一种分子量为20 kDa的蛋白质，具有针对细胞膜的成孔能力，其功能最近引起了极大关注。在本研究中，其编码序列被克隆为带有His标签的融合蛋白，以便于简单生产和快速纯化。
使用携带引物进行PCR扩增后，将基因片段克隆到 。 使用构建的载体进行表达，并通过SDS-PAGE验证毒素表达。采用Ni-NTA琼脂糖亲和层析进行一步纯化。为检测纯化毒素的功能，进行了红细胞溶血试验。
结果表明，FraC编码基因成功克隆于 和 限制酶切位点之间，并通过亲和层析纯化。光密度分析表明纯度约为97%。溶血试验表明，纯化的FraC对红细胞具有显著的裂解活性，在浓度为6.25 nM时几乎裂解了50%的细胞。
结果表明，纯化的毒素不仅在表达和纯化过程中保留了其活性，而且在较低浓度下也发挥了其功能，以至于即使在0.09 nM时也显示出溶血作用。
