摘要
利用野生东亚钳蝎毒腺构建cDNA文库,并通过序列比对确定了一条全新的Na^+通道长链毒素BmkNaTx12并克隆出其全长cDNA序列。序列比对显示BmkNaTx12的序列与墨西哥毒蝎的β-毒素Cn12具有较高的结构相似性,通过同源模型构建并选取打分最高的模型得到BmkNaTx12的预测结构,经分子动力学优化发现蛋白结构在300 ns趋于稳定,而蛋白20、35、52个氨基酸处的波动很可能由于这些氨基酸所处位置在loop区域导致。进一步构建了BmkNaTx12-Fc融合蛋白重组表达系统,通过Western blot技术确定重组蛋白最佳表达时间,并在HEK293细胞中成功表达出BmkNaTx12-Fc融合蛋白。经蛋白A亲和色谱纯化后获得的重组融合蛋白经SDS-PAGE凝胶电泳及高效液相鉴定后纯度高达95%。此外,全细胞膜片钳实验结果显示:BmkNaTx12-Fc在1μmol/L浓度下能增大20%Nav1.7峰电流。研究结果为后续的生物学功能研究奠定了理论基础。
A cDNA library was constructed from glands of wild Buthus martensii Karsch scorpion. A new sodium channel long-chain toxin BmkNaTx12 was identified by sequence alignment, and its full-length cDNA sequence was cloned. Sequence alignment showed that the sequence of BmkNaTx12 had high structural similarity with the β-toxin Cn12 from Mexican Centruroides noxius scorpion. The predicted structure of BmkNaTx12 was obtained by homologous model construction and the highest scoring model was selected. The protein structure was found to be stable at 300 ns by molecular dynamics optimization, and fluctuations at amino acids 20, 35, and 52 are most likely due to the location of these amino acids in the loop region. Then, an expression system of recombinant BmkNaTx12-Fc fusion protein was constructed. The optimal expression time of recombinant protein was determined by Western blot and the fusion protein was successfully expressed in HEK293 cells. The purity of recombinant fusion protein which was obtained by protein A affinity chromatography was up to 95% by SDS-PAGE gel electrophoresis and HPLC. In addition, the whole-cell patch-clamp assay results suggest that 1 μmol/L BmkNaTx12-Fc can increase 20% Nav1. 7 peak current. These results laid a foundation for the further study of biological function.
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2017年第2期220-226,共7页
Journal of China Pharmaceutical University
基金
2015年度公益性行业科研专项经费项目(No.201507002)
国家自然科学基金资助项目(No.81622048
No.81473377
No.81573837)
江苏省自然科学基金资助项目(No.BK20140049)~~
关键词
东亚钳蝎
蝎毒素
基因克隆
表达
Na^+通道
Buthus martensii Karsch
scorpion toxin
gene cloning
expression
Na^＋ channel
