Szynol A, de Soet J J, Sieben-van Tuyl E, Bos J W, Frenken L G
Department of Periodontology, Section Oral Microbiology, Academic Centre for Dentistry, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
Antimicrob Agents Chemother. 2004 Sep;48(9):3390-5. doi: 10.1128/AAC.48.9.3390-3395.2004.
Enzymes such as lactoperoxidase and glucose oxidase (GOx) are used as antimicrobial agents in oral care products. Their low specificities and substantiveness can be reduced by covalent coupling of antimicrobial molecules to antibodies. Variable domains (V(HH)) derived from llama heavy-chain antibodies are particularly suited for such an approach. The antibodies are composed solely of heavy-chain dimers; therefore, production of active fusion proteins by using molecular biology-based techniques is less complicated than production by use of conventional antibodies. In this study, a fusion protein consisting of V(HH) and GOx was constructed and expressed by Saccharomyces cerevisiae. A llama was immunized with Streptococcus mutans strain HG982. Subsequently, B lymphocytes were isolated and cDNA fragments encoding the V(HH) fragments were obtained by reverse transcription-PCR. After construction of a V(HH) library in Escherichia coli and screening of the library against mutans group streptococci and Streptococcus sanguinis strains, we found two V(HH) fragments with high specificities for S. mutans strains. A GOx gene was linked to the two V(HH) genes and cloned into S. cerevisiae yeasts. The yeasts expressed and secreted the recombinant proteins into the growth medium. The test of binding of fusion proteins to oral bacteria through their V(HH) fragments showed that S. mutans had been specifically targeted by GOx-S120, one of the fusion protein constructs. A low concentration of the fusion protein was also able to selectively kill S. mutans within 20 min in the presence of lactoperoxidase and potassium iodide. These findings demonstrate that the fusion protein GOx-V(HH) is potentially valuable in the selective killing of target bacteria such as S. mutans.
诸如乳过氧化物酶和葡萄糖氧化酶（GOx）等酶类被用作口腔护理产品中的抗菌剂。通过将抗菌分子与抗体共价偶联，可以降低它们的低特异性和亲和性。源自羊驼重链抗体的可变区（V(HH)）特别适合这种方法。这些抗体仅由重链二聚体组成；因此，使用基于分子生物学的技术生产活性融合蛋白比使用传统抗体生产要简单。在本研究中，构建了一种由V(HH)和GOx组成的融合蛋白，并由酿酒酵母表达。用变形链球菌HG982菌株免疫一只羊驼。随后，分离出B淋巴细胞，并通过逆转录聚合酶链反应获得编码V(HH)片段的cDNA片段。在大肠杆菌中构建V(HH)文库，并针对变形链球菌群和血链球菌菌株筛选该文库后，我们发现了两个对变形链球菌菌株具有高特异性的V(HH)片段。将一个GOx基因与这两个V(HH)基因连接，并克隆到酿酒酵母中。酵母将重组蛋白表达并分泌到生长培养基中。通过融合蛋白的V(HH)片段与口腔细菌结合的试验表明，融合蛋白构建体之一GOx-S120能够特异性靶向变形链球菌。在存在乳过氧化物酶和碘化钾的情况下，低浓度的融合蛋白也能够在20分钟内选择性杀死变形链球菌。这些发现表明，融合蛋白GOx-V(HH)在选择性杀死诸如变形链球菌等目标细菌方面具有潜在价值。
