Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China.
PLoS One. 2013 May 23;8(5):e64106. doi: 10.1371/journal.pone.0064106. Print 2013.
Insect prophenoloxidase (PPO) is essential for physiological functions such as melanization of invading pathogens, wound healing and cuticle sclerotization. The insect PPO activation pathway is well understood. However, it is not very clear how PPO is released from hemocytes and how PPO takes part in cellular immunity. To begin to assess this, three Drosophila melanogaster PPO genes were separately fused with GFP at the C-terminus (rPPO-GFP) and were over-expressed in S2 cells. The results of staining and morphological observation show that rPPO-GFP expressed in S2 cells has green fluorescence and enzyme activity if Cu(2+) was added during transfection. Each rPPO-GFP has similar properties as the corresponding rPPO. However, cells with rPPO-GFP over-expressed are easier to trace without PO activation and staining. Further experiments show that rPPO1-GFP is cleaved and activated by Drosophila serine protease, and rPPO1-GFP binds to Micrococcus luteus and Beauveria bassiana spores as silkworm plasma PPO. The above research indicates that the GFP-tag has no influence on the fusion enzyme activation and PPO-involved innate immunity action in vitro. Thus, rPPO-GFP may be a convenient tool for innate immunity study in the future if it can be expressed in vivo.
昆虫酚氧化酶（PPO）对于生理功能至关重要，如入侵病原体的黑化、伤口愈合和表皮硬化。昆虫 PPO 激活途径已被充分理解。然而，PPO 如何从血细胞中释放以及 PPO 如何参与细胞免疫尚不清楚。为了开始评估这一点，分别将三个果蝇 PPO 基因与 GFP 在 C 端融合（rPPO-GFP），并在 S2 细胞中过表达。染色和形态观察的结果表明，在转染过程中添加 Cu(2+)时，在 S2 细胞中表达的 rPPO-GFP 具有绿色荧光和酶活性。每个 rPPO-GFP 都具有与相应 rPPO 相似的性质。然而，过表达 rPPO-GFP 的细胞在没有 PO 激活和染色的情况下更容易追踪。进一步的实验表明，rPPO1-GFP 被果蝇丝氨酸蛋白酶切割和激活，并且 rPPO1-GFP 与金黄色葡萄球菌和球孢白僵菌孢子结合，就像家蚕血浆 PPO 一样。上述研究表明，GFP 标签在体外对融合酶的激活和 PPO 参与固有免疫没有影响。因此，如果 rPPO-GFP 能够在体内表达，它可能成为未来固有免疫研究的一种便捷工具。
