Instituto de Agrobiotecnología, Universidad Pública de Navarra-CSIC-Gobierno de Navarra, Campus Arrosadía, 31006 Pamplona, Spain,
Planta. 2010 Mar;231(4):977-90. doi: 10.1007/s00425-010-1102-4. Epub 2010 Jan 28.
We previously showed that recombinant extra domain A from fibronectin (EDA) purified from Escherichia coli was able to bind to toll-like receptor 4 (TLR4) and stimulate production of proinflammatory cytokines by dendritic cells. Because EDA could be used as an adjuvant for vaccine development, we aimed to express it from the tobacco plastome, a promising strategy in molecular farming. To optimize the amount of recombinant EDA (rEDA) in tobacco leaves, different downstream sequences were evaluated as potential fusion tags. Plants generated by tobacco plastid transformation accumulated rEDA at levels up to 2% of the total cellular protein (equivalent to approximately 0.3 mg/g fresh weight) when translationally fused to the first 15 amino acids of green fluorescence protein (GFP). The recombinant adjuvant could be purified from tobacco leaves using a simple procedure, involving ammonium sulfate precipitation and anion exchange chromatography. Purified protein was able to induce production of tumour necrosis factor-alpha (TNF-alpha) either by bone marrow-derived dendritic cells or THP-1 monocytes. The rEDA produced in tobacco leaves was also able to induce upregulation of CD54 and CD86 maturation markers on dendritic cells, suggesting that the rEDA retains the proinflammatory properties of the EDA produced in E. coli and thus could be used as an adjuvant in vaccination against infectious agents and cancer. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of this protein vaccine adjuvant.
我们之前曾表明，从大肠杆菌中纯化的纤维连接蛋白（FN）的额外结构域 A（EDA）重组蛋白能够与 Toll 样受体 4（TLR4）结合，并刺激树突状细胞产生促炎细胞因子。由于 EDA 可用作疫苗开发的佐剂，我们旨在从烟草质体中表达它，这是分子农业中很有前途的策略。为了优化烟草叶片中重组 EDA（rEDA）的含量，我们评估了不同的下游序列作为潜在的融合标签。通过烟草质体转化产生的植物，当与 GFP 的前 15 个氨基酸翻译融合时，rEDA 可积累到总细胞蛋白的 2%（相当于约 0.3 mg/g 鲜重）。重组佐剂可通过简单的程序从烟草叶片中纯化，包括硫酸铵沉淀和阴离子交换层析。纯化的蛋白能够诱导骨髓来源的树突状细胞或 THP-1 单核细胞产生肿瘤坏死因子-α（TNF-α）。从烟草叶片中产生的 rEDA 也能够诱导树突状细胞上 CD54 和 CD86 成熟标志物的上调，这表明 rEDA 保留了大肠杆菌产生的 EDA 的促炎特性，因此可用于针对感染因子和癌症的疫苗接种佐剂。综上所述，这些结果表明叶绿体是表达这种蛋白疫苗佐剂的有吸引力的生产载体。
