美洲拟鲽抗菌肽Pleurocidin在大肠杆菌中的高效分泌表达及优化

Expression of Pleurocidin from winter flounder in Escherichia coli and optimization of culture conditions

摘要:

为在大肠杆菌中分泌表达Pleurocidin，并提高融合蛋白的分泌效率，将Pleurocidin基因和Cherry DNA序列通过平末端连接融合，再将融合基因整合到pET22b (+) 载体中，转化大肠杆菌BL21 (DE3)；乳糖诱导表达。成功构建含pET22b (+)-CP重组质粒的基因工程菌，用乳糖诱导获得高效表达。在诱导16 h时加入甘氨酸可以显著提高融合蛋白Cherry-Pleurocidin的分泌效率。用稀盐酸水解融合蛋白的酸敏感位点，再进一步分离纯化即得到r-Pleurocidin，其对大肠杆菌DH5α和枯草芽孢杆菌BS168具有明显的抑菌活性。结果表明成功构建了高效表达Pleurocidin的大肠杆菌基因工程菌，获得有活性的r-Pleurocidin。

Abstract:

To express Pleurocidin in Escherichia coli and to enhance the secretory efficiency of the fusion protein, the gene encoding Pleurocidin was ligated with Cherry DNA sequence via blunt-end ligation. Then this fusion gene was cloned into pET22b (+) vector and the recombinant plasmid was transformed into E. coli BL21 (DE3). Lactose was used to induce expression of fusion protein. The recombinant plasmid pET22b (+) -CP was successfully constructed and high-level expression of fusion protein was induced with lactose. Statistics showed that addition of glycine after 16 h of induction significantly enhanced the secretory efficiency of the fusion protein. After hydrolysis of the fusion protein by diluted hydrochloric acid and some further purification steps, r-Pleurocidin was obtained with antibacterial activity against E. coli DH5α and Bacillus subtilis BS168. In conclusion, the fusion protein was expressed in E. coli and biologically active r-Pleurocidin was obtained after hydrochloric acid cleavage and purification.