Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system
Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system
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DOI：
10.1096/fj.05-5134fje
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发表时间：
2006-05-01
期刊：
影响因子：
4.8
通讯作者：
Daniell, Henry
中科院分区：
文献类型：
--
作者：
Limaye, Arati;Koya, Vijay;Daniell, Henry
口服递送在植物细胞中表达的生物药物蛋白质应降低其生产、纯化、加工、冷藏、运输和递送的成本。然而，完整蛋白质的肠道吸收不良是一个主要挑战。为了克服这一限制，我们在这里调查的概念受体介导的叶绿体表达的外源蛋白的口服递送。因此，跨粘膜载体霍乱毒素B亚基和绿色荧光蛋白（CTB-GFP），分离的弗林蛋白酶切割位点，通过烟草叶绿体基因组表达。聚合酶链反应（PCR）和Southern印迹分析证实了位点特异性转基因整合和同质性。免疫印迹分析和ELISA证实CTB-GFP的单体和五聚体形式的表达，高达总可溶性蛋白的21.3%。体外弗林蛋白酶切割测定证实了工程化弗林蛋白酶切割位点的完整性，GM（1）结合测定证实了CTB-GFP五聚体的功能性。对小鼠口服CTB-GFP表达叶材料后，在荧光和免疫组织化学研究中在小鼠肠粘膜、肝和脾中观察到GFP，而CTB保留在肠细胞中。受体介导的外源蛋白质口服递送到循环系统的这一报告为低成本生产和递送人类治疗蛋白质打开了大门。
Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM(1) binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins.