School of Life Sciences, Shandong University, Jinan, Shandong 250100, China.
Fish Shellfish Immunol. 2010 May-Jun;28(5-6):942-51. doi: 10.1016/j.fsi.2010.02.011. Epub 2010 Feb 17.
Three Kazal-type serine proteinase inhibitors, hcPcSPI2, hpPcSPI3, and hpPcSPI4, with complete cDNA sequences, were identified from a cDNA library of the red swamp crayfish, Procambarus clarkii. Semi-quantitative RT-PCR shows that hcPcSPI2 exists mainly in hemocytes while both hpPcSPI3 and hpPcSPI4 were detected in the hepatopancreas and the heart. Homology comparison and phylogenic analysis indicate that hpPcSPI3 and hpPcSPI4 shared high identity and formed the same group, and both of them were different from other hepatopancreas type inhibitors in crustaceans forming a large group, while hcPcSPI2 as well as other hemocyte type inhibitors belonged to another cluster. In addition, the temporal expression profiles of these three inhibitors were studied with quantitative real-time PCR and the results suggest that hcPcSPI2 and hpPcSPI3 are likely to be involved in antiviral immune response, and all these three inhibitors respond to Vibrio anguillarum challenge in different degrees. Further study was done on hcPcSPI2. Western blot demonstrates that hcPcSPI2 only exists in semigranular cells. Besides, after V. anguillarum challenge, the hcPcSPI2 protein could also be detected in cell-free hemolymph. Subsequently, the biochemical characteristics and bacteriostatic activity of hcPcSPI2 were assayed. The results indicate that hcPcSPI2 shows weak inhibitory activity against subtilisin A and trypsin, and may trigger bacteriostatic activity towards Bacillus subtilis and Bacillus thuringiensis, possessing MIC(50) of 30.4 and 25.0 microM, respectively. These studies reveal that hcPcSPI2 may also play an important role in the anti-bacterial immunity of the crayfish.
从克氏原螯虾（Procambarus clarkii） cDNA 文库中鉴定出三种完整 cDNA 序列的 Kazal 型丝氨酸蛋白酶抑制剂，hcPcSPI2、hpPcSPI3 和 hpPcSPI4。半定量 RT-PCR 显示 hcPcSPI2 主要存在于血细胞中，而 hpPcSPI3 和 hpPcSPI4 则在肝胰腺和心脏中被检测到。同源性比较和系统发育分析表明，hpPcSPI3 和 hpPcSPI4 具有高度的同源性，形成同一组，与甲壳动物其他肝胰腺型抑制剂不同，形成一个大组，而 hcPcSPI2 以及其他血细胞型抑制剂则属于另一组。此外，还通过定量实时 PCR 研究了这三种抑制剂的时间表达谱，结果表明 hcPcSPI2 和 hpPcSPI3 可能参与抗病毒免疫反应，这三种抑制剂对鳗弧菌的挑战都有不同程度的反应。进一步研究了 hcPcSPI2。Western blot 表明 hcPcSPI2 仅存在于半颗粒细胞中。此外，在鳗弧菌攻击后，hcPcSPI2 蛋白也可以在无细胞的血淋巴中检测到。随后，测定了 hcPcSPI2 的生化特性和抑菌活性。结果表明，hcPcSPI2 对枯草杆菌蛋白酶 A 和胰蛋白酶表现出较弱的抑制活性，可能对枯草芽孢杆菌和苏云金芽孢杆菌触发抑菌活性，MIC(50)分别为 30.4 和 25.0 μM。这些研究表明，hcPcSPI2 可能在螯虾的抗细菌免疫中也发挥重要作用。
