基于枯草芽孢杆菌重组菌株构建技术制备海洋源金属硫蛋白

Preparation of marine sources metallothionein based on construction of recombinant strain of Bacillus subtilis

为构建安全高效表达海洋源金属硫蛋白的益生菌重组菌株,以海洋源褶牡蛎内脏团为模板,PCR克隆得到褶牡蛎金属硫蛋白基因(Op-MT),大小为324 bp,经pMD19-T载体亚克隆到融合表达载体pHT43-SUMO中,然后通过电转化法将重组质粒转入八种蛋白酶缺陷的宿主枯草芽孢杆菌(Bacillus subtilis)WB800N中。SDS-PAGE电泳显示诱导表达的融合蛋白SUMO-Op-MT分子量约为23 kD,与预期一致,说明可溶性融合蛋白SUMO-Op-MT在宿主菌中得到了表达,且通过Western blotting进一步得到验证。同时,HPLC检测重组枯草芽孢杆菌发酵液,结果也表明重组枯草芽孢杆菌成功表达了可溶性目的蛋白Op-MT。通过对重组工程菌中MT活性检测发现,其对重金属镉耐受力增强,说明重组工程菌成功表达出有活性的金属硫蛋白。这为以后开发天然、高效海洋源重金属解毒剂提供理论基础。

In order to facilitate marine sources metallothionein,safe and high-level expression system as an efficient tool in Bacillus subtilis was reconstructed in this study.The metallothionein(MT)gene was obtained by PCR amplification using Ostrea plicatula genome DNA as a template and sub-cloned into the expression plasmid pHT43 via pMD19-Tvector.Then the plasmid was electro-transformed into B.subtilis WB800N.The molecular weight of expressed fusion protein SUMO-Op-MT was about 23 kD,which was confirmed by Western blotting.This showed that the soluble fusion protein SUMO-Op-MT was expressed in the host bacteria.At the same time,the fermentation broth of recombinant Bacillus subtilis was detected by HPLC,and the results also showed that the soluble protein Op-MT was expressed in recombinant Bacillus subtilis successfully.Furthermore,through the detection of MT activity in recombinant bacteria,it was found that recombinant bacteria s tolerance to heavy metal cadmium was increased,indicating that active MT was successfully expressed in recombinant bacteria.It provides a theoretical basis for the future development of heavy metal antidote of natural and efficient marine sources.