PDF(Pubmed)
Abstract：
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.
摘要：
昆虫酚氧化酶原（PPO）是必不可少的生理功能，如黑化的入侵病原体，伤口愈合和角质层硬化。昆虫PPO激活途径是很好理解的。然而,目前尚不清楚PPO是如何从血细胞中释放的，以及PPO是如何参与细胞免疫的。为了开始评估这一点，三个果蝇PPO基因分别在C端与GFP融合（rPPO-GFP），并在S2细胞中过表达。染色和形态学观察结果表明，如果在转染过程中添加Cu（2），则在S2细胞中表达的rPPO-GFP具有绿色荧光和酶活性。每个rPPO-GFP具有与相应的rPPO相似的性质。然而,rPPO-GFP过表达的细胞更容易追踪，而无需PO激活和染色。进一步的实验表明，rPPO1-GFP被果蝇丝氨酸蛋白酶切割和激活，rPPO1-GFP作为家蚕血浆PPO与藤黄微球菌和球孢白僵菌孢子结合。以上研究表明，GFP标签在体外对融合酶的激活和PPO参与的先天免疫作用没有影响。因此，如果rPPO-GFP可以在体内表达，则可能是将来进行先天免疫研究的便捷工具。
