何永吉,刘进平,李颖君,王兰.华溪蟹功能性重组金属硫蛋白的表达及抗血清制备[J].环境科学学报,2014,34(9):2434-2439
华溪蟹功能性重组金属硫蛋白的表达及抗血清制备
- Expression of functional metallothionein of freshwater crab (Sinopotamon henanense) in E. coli and preparation of its antiserum
- 基金项目：国家自然科学基金（No. 30970361）；高等学校博士学科点专项科研基金（No. 20111401110010）；山西省自然科学基金（No.2010011043-2）
- 摘要：采用SUMO融合表达系统表达河南华溪蟹金属硫蛋白（Metallothionein，MT），经Ni 离子螯合柱分离纯化后，用SDS-PAGE、紫外光谱扫描鉴定，并对重组MT的金属结合及清除自由基能力进行了检测.同时，利用纯化的融合蛋白SUMO-MT免疫BALB/c小鼠制备抗血清，间接ELISA检测该抗血清的效价，Western blot和免疫组化染色检测其特异性.结果表明，河南华溪蟹MT以可溶形式获得表达，经金属螯合层析纯化后分析仍具有金属结合特性和清除羟自由基的生物学功能.免疫BALB/c小鼠后获得特异性的抗MT抗血清，该抗血清具有较高的效价和良好的特异性，可识别组织MT，用于免疫组化分析.
- Abstract：To produce functional metallothionein (MT) of freshwater crab (Sinopotamon henanense) and its antiserum, the gene for the crab MT was over-expressed as fusions with SUMO in Escherichia coli and purified by affinity chromatography. The purified MT was identified by SDS-PAGE and UV absorbance spectrometry analysis. Chemical and functional characteristics of the recombinant MT were investigated. Subsequently, the BALB/c mice were immunized with the purified MT and the resulted antiserum was analyzed by ELISA, Western blotting and immunohistochemical stainting. Results showed that the high purity MT was obtained by SUMO fusion protein system and exhibited intact metal binding ability and hydroxyl radical scavenging ability. The antiserum of high titer against MT of S. henanense was prepared in mice and was proven to have a good specificity by Western bloting and immunohistochemical stainting. The present study could help to elucidate the mechanism of how the crab MT is effective in heavy metal detoxification and provide a scientific basis for development of an ELISA for determination of MT.
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