β-防御素130在大肠杆菌中的串联表达、纯化及生物活性分析

Tandem expression, purification and biological activity of recombinant multimeric β-defensin130 in Escherichia coli

摘要:

为了研究抗菌肽β-防御素130的生物学活性和实现大规模制备，通过改良其分子结构，构建表达载体pET28a-3×β-defensin130，利用大肠杆菌BL21 (DE3) 作为宿主细胞诱导表达后为水溶性蛋白。对纯化后抗菌肽进行抑菌实验、稳定性实验、MTT实验和溶血性实验确定其生物活性。最终成功制备出25 kDa的重组蛋白，对金黄色葡萄球菌 (ATCC 25923)(45 μg/mL) 和单增李斯特菌 (ATCC 221633) (80 μg/mL) 等革兰氏阴性和阳性菌都表现出极强的抗菌活性，且其抗菌活性不受温度、pH值和蛋白酶消化等影响，MTT细胞毒性实验显示其对HEK293细胞无毒性且对兔源红细胞具有极低的溶血性。这将为新型抗菌肽的开发提供理论基础并推动抗生素替代产业快速发展。

关键词:防御素，抗菌肽，原核表达，蛋白纯化，抗菌活性

Abstract:

To improve and broaden the antimicrobial activity of β-defensin130, 3 copies of β-defensin130 encoding sequences were synthesized and cloned into pET28a (+) expression vector, and expressed in Escherichia coli BL21 (DE3) as a 25 kDa soluble protein. The affinity purified 3×β-defensin 130 displayed antimicrobial activity against not only Gram-positive strains including Staphylococcus aureus (ATCC 25923) (45 μg/mL) and Listeria monocytogenes (ATCC 221633) (80 μg/mL) but also Gram-negative strains. Furthermore, the antimicrobial activity of β-defensin130 was not affected by temperature, pH and proteinase digestion. In addition, E. coli-derived 3×β-defensin130 was not toxic to HEK 293 cells and showed a relatively low hemolytic activity against rabbit erythrocytes. Our study proves 3×β-defensin130 expressed in E. coli is stable, non-cytotoxic and low-hemolytic active with great potential as alternative antibiotics.

Key words:defensin, antimicrobial peptide, prokaryotic expression, protein purification, antimicrobial activity