B型肉毒毒素保护性抗原Hc在大肠杆菌中的可溶性高表达

High-Level and Soluble Expressions of Botulinum Neurotoxin Serotype B Protective Antigen Hc Domain in Escherichia coli

目的:通过序列优化及表达条件改进,在大肠杆菌中高效可溶性表达B型肉毒毒素保护性抗原Hc(Bont/B-Hc)。方法:对Bont/B-Hc基因片段优化,用大肠杆菌常用密码子替换稀有密码子,并将(G+C)含量由76.2%降至56.3%;人工合成多条具有重叠互补序列的寡核苷酸片段,采用重叠延伸PCR方法获得了Bont/B-Hc的全长基因,并构建了原核可溶性表达载体;将经酶切和测序鉴定正确的重组质粒转化大肠杆菌BL21(DE3)感受态细胞,用IPTG诱导Bont/B-Hc的表达并进行纯化及Western印迹鉴定。结果和结论:目的蛋白在大肠杆菌BL21(DE3)中获得了可溶性高表达,占菌体裂解液上清总蛋白的26.7%,表达量达到30mg/L,是目前国内外已知表达的最高水平;经Ni柱一步纯化后,目的蛋白纯度可达到80.3%,为肉毒毒素中和抗体的制备及亚单位疫苗的研究奠定了基础。

Objective： To explore the high-level and soluble expression of the reconstructed protective antigen He fragment of botulinum neurotoxin serotype B （Bont/B-Hc） in E.coli by optimizing the sequence and expression conditions. Methods： The gene encoding Bont/B-Hc was cloned and optimized by replacing rare codons with frequent ones in E.coli and the content of GC pairs decreased from 76.2% to 56.3%. The reconstructed gene was synthesized by overlapping PCR and cloned into prokaryotic soluble expression vector. The recombinant plasmid was then introduced into E.coli BL21（DE3）. Bont/B-Hc was expressed, purified and identified by Western-blot. Results and conclusions： Reconstructed He gene was expressed in E.coli BL21（DE3） in soluble form which covered 26.7% total proteins in the supematant of sonicated bacteria. It can be high as 30 mg/L and there was no reports about higher expression before. When purified with Ni column, He covered 80.3% total proteins. This will be an important basis for the produce of neutralizing antibodies and sub-unit vaccines of butulinum neurotoxin serotype B.