β-defensin 2 synthesized by a cell-free protein synthesis system and encapsulated in liposomes inhibits adhesion of Porphyromonas gingivalis to oral epithelial cells
β-defensin 2 synthesized by a cell-free protein synthesis system and encapsulated in liposomes inhibits adhesion of Porphyromonas gingivalis to oral epithelial cells
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DOI：
10.1007/s10266-023-00789-x
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发表时间：
2023-02-06
期刊：
影响因子：
2.5
通讯作者：
Shinohara，Yasuo
中科院分区：
文献类型：
--
作者：
Hiroshima，Yuka;Kido，Jun-ichi;Shinohara，Yasuo
β-防御素2（β-defensin 2，BD-2）是一种抗菌肽，由口腔上皮细胞表达，在口腔天然免疫中起重要作用。已经研究了无细胞蛋白质合成（CFPS）系统用于合成各种蛋白质，然而，通过CFPS系统合成BD-2尚未被广泛探索。脂质体已被开发为用于药物递送的工具。将脂质体包封的AMP递送至口腔上皮可能有助于预防口腔感染性疾病。在本研究中，我们研究了BD-2蛋白的抗微生物活性，使用CFPS系统人工合成并封装在脂质体中。利用模板DNA和重组CFPS系统人工合成BD-2蛋白，并通过western blotting进行鉴定。以蛋黄中的1，2-二油酰-sn-甘油-3-磷酸胆碱和3-sn-磷脂酰胆碱为原料制备双层脂质体。将人工合成的BD-2包封在脂质体中，通过超滤收集，并通过蛋白质印迹检测。人口腔上皮细胞与脂质体包封的BD-2一起培养，并且具有合成的BD-2的培养物的细胞裂解物中BD-2的浓度高于对照培养物的浓度。通过牙龈卟啉单胞菌对口腔上皮细胞的粘附试验研究了合成的BD-2的抗菌活性。人工合成的BD-2及其脂质体对P. gingivalisto口腔上皮细胞。这些结果表明，人工合成的BD-2和脂质体包封的BD-2显示抗菌活性，并可能在牙周疾病的口腔保健中发挥作用。
β-defensin 2 (BD-2), an antimicrobial peptide (AMP), is expressed by oral epithelial cells and plays an important role in innate immunity of the oral cavity. Cell-free protein synthesis (CFPS) systems have been studied for the synthesis of various proteins, however, the synthesis of BD-2 by a CFPS system has not been extensively explored. Liposomes have been developed as tools for drug delivery. A delivery of liposome-encapsulated AMP to oral epithelium may be useful to prevent oral infectious diseases. In the present study, we investigated the antimicrobial activity of the BD-2 protein, artificially synthesized using a CFPS system and encapsulated in liposomes. BD-2 protein was artificially synthesized using template DNA and a reconstituted CFPS system and was identified by western blotting. Bilayer liposomes were prepared using 1,2-dioleoyl-sn-glycero-3-phospho-choline and 3-sn-phosphatidylcholine from egg yolk. The artificially synthesized BD-2 was encapsulated in liposomes, collected by ultrafiltration, and detected by western blotting. Human oral epithelial cells were cultured with the liposome-encapsulated BD-2 and the concentration of BD-2 in the cell lysate of the culture with the synthesized BD-2 was higher than that of the control cultures. The antimicrobial activity of the synthesized BD-2 was investigated by an adhesion assay ofPorphyromonas gingivalisto oral epithelial cells. The artificially synthesized BD-2 and its liposome significantly inhibited adhesion ofP. gingivalisto oral epithelial cells. These results suggest that artificially synthesized BD-2 and liposome-encapsulated BD-2 show antimicrobial activity and can potentially play a role in oral healthcare for periodontal diseases.