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目的克隆B型肉毒毒素轻链Bont-B基因,使其在大肠杆菌内表达,并对表达的重组蛋白进行纯化。方法设计并人工合成Bont-B基因,克隆入表达载体pMD19-T中,构建质粒pMD19-T-Bont-B,经酶切后与pET-28a载体连接,构建重组表达质粒pET-28a-Bont-B,将重组表达质粒转化感受态大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE及Western blot分析后,经金属螯合层析Cu2+柱进行纯化,纯化产物经SDS-PAGE分析纯度。结果重组表达质粒pET-28a-Bont-B经PCR、双酶切及测序鉴定,证明构建正确;表达的重组蛋白相对分子质量约50 000,主要以可溶性形式表达,表达量约占菌体总蛋白的6%;纯化后的重组蛋白纯度可达90%以上。结论已成功克隆并在大肠杆菌BL21(DE3)中原核表达了Bont-B轻链基因,为制备抗Bont-B轻链单克隆抗体及研究肉毒中毒机制和治疗奠定了基础。
Objective To clone the Bont-B gene of botulinum toxin type B, make it express in E. coli and purify the expressed recombinant protein. Methods The Bont-B gene was designed and synthesized and cloned into the expression vector pMD19-T. The plasmid pMD19-T-Bont-B was constructed and ligated with pET-28a vector to construct the recombinant plasmid pET-28a- B, the recombinant expression plasmid was transformed into competent E. coli BL21 (DE3), IPTG induced expression. The expressed product was analyzed by SDS-PAGE and Western blot and purified by metal chelate chromatography on Cu2 + column. The purity of the purified product was analyzed by SDS-PAGE. Results The recombinant plasmid pET-28a-Bont-B was confirmed by PCR, restriction enzyme digestion and sequencing. The recombinant plasmid pET-28a-Bont-B was correctly constructed. The recombinant protein was expressed in soluble form and expressed in soluble form, Of the 6% purity of purified recombinant protein up to 90%. Conclusion The Bont-B light chain gene was successfully cloned and expressed in E.coli BL21 (DE3) prokaryotic cells, which laid the foundation for the preparation of monoclonal antibodies against Bont-B light chain and the study on the mechanism and treatment of botulism.