抗菌肽Mytimacin-4在大肠杆菌中的高效表达及其活性鉴定

High-level Expression of Antimicrobial Peptide Mytimacin-4 in Escherichia coli and Identification of Its Activity

摘要:

已有研究表明，Mytilus galloprovincialis来源的macin-4对革兰氏阳性和革兰氏阴性菌均有较强的抑菌活性，但关于其生产的研究甚少，限制了其后续的产品开发及应用。该研究通过对mytimacin-4进行大肠杆菌密码子优化并构建在pET-28a表达载体上，将构建成功的pET-28a-mytimacin-4分别和含不同组合分子伴侣的表达载体pG-KJE8、pKJE7、pGro7、pG-Tf2和pTf16共转化到大肠杆菌BL21（DE3）表达宿主，进行诱导表达及活性鉴定。结果表明，共表达分子伴侣可显著提高Mytimacin-4在大肠杆菌中的可溶性表达，其中，重组菌株BL21(DE3)/pET-28a-mn4/pG-KJE8优化后的产量可达200~400 mg/L，是目前已报道的最高产量。表达产物经金属离子亲和层析，得到的纯化产物Mytimacin-4对金黄色葡萄球菌、大肠杆菌和副溶血弧菌均有较强的抑菌活性。该研究构建了一株高效表达Mytimacin-4的工程菌株，为Mytimacin-4在兽药和水产饲料领域的应用提供了技术支撑。

Abstract:

Mytimacin-4 from Mytilus galloprovincialis has strong bacteriostatic activity against gram-positive and gram-negative bacteria. However, the production of mytimacin-4 has been rarely studied, which limits its subsequent product development and application. In this study, the gene sequence of mytimacin-4 was codon optimized in Escherichia coli and constructed in the expression vector pET-28a. Then, pET-28a-mytimacin-4 and each of the expression vectors containing different combinations of molecular chaperones (pG-KJE8, pKJE7, pGro7, pG-Tf2, and pTf16) were co-transformed into E. coli BL21 (DE3) for induced expression and activity identification. The results showed that the co-expression of molecular chaperones significantly improved the soluble expression of mytimacin-4 in Escherichia coli. Notably, the optimized yields of the recombinant strain BL21(DE3)/pET-28a-mn4/pG-KJE8 ranged from 200 to 400 mg/L, the highest reported. Purified mytimacin-4 was obtained by metal-ion affinity chromatography product of the expression products. The purified mytimacin-4 showed strong inhibitory activity against Staphylococcus aureus, E. coli, and Vibrio parahaemolyticus. In this study, an engineering strain with high expression of mytimacin-4 was constructed, which provided technical support for applying mytimacin-4 in veterinary medicine and aquatic feed.