Wongprasert Kanokpan, Sangsuriya Pakkakul, Phongdara Amornrat, Senapin Saengchan
Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand.
J Biotechnol. 2007 Aug 1;131(1):9-19. doi: 10.1016/j.jbiotec.2007.05.032. Epub 2007 Jun 6.
A black tiger shrimp (Penaeus monodon) caspase cDNA homologue (PmCasp) has been identified from a hemocyte library using a previously identified caspase homologue from the banana shrimp (Penaeus merguiensis) as a probe. The full-length PmCasp was 1202bp with a 954bp open reading frame, encoding 317 amino acids. The deduced protein contained a potential active site (QACRG pentapeptide) conserved in most caspases. It had 83% identity with caspase of P. merguiensis and 30% identity with drICE protein of Drosophila melanogaster, and it exhibited caspase-3 activity in vitro. PmCasp was cloned and expressed in Escherichia coli and a rabbit polyclonal antiserum was produced. In Western blots, the antiserum reacted with purified recombinant PmCasp and with lysates of E. coli containing the expressed plasmid. In crude protein extracts from normal shrimp, the antiserum reacted with 36 and 26kDa bands likely to correspond to inactive pro-caspase and its proteolytic intermediate form, respectively. PmCasp expression was measured in normal shrimp and in white spot syndrome virus (WSSV)-infected shrimp at 24 and 48h post-injection (p.i.) by semi-quantitative RT-PCR, Western blot analysis, and immunohistochemistry. Semi-quantitative RT-PCR analysis revealed up-regulation of PmCasp at 48h p.i. and expression remained high up to the moribund state. These results were supported by Western blot analysis showing increased PmCasp protein levels at 24 and 48h p.i. when compared to normal control shrimp. Immunohistochemical analysis of gills from the WSSV-infected shrimp revealed immunoreactivity localized in the cytoplasm of both normal and apparently apoptotic cells. In summary, a caspase-3 like gene is conserved in P. monodon and is up-regulated after WSSV infection.
利用先前从斑节对虾（Penaeus merguiensis）中鉴定出的半胱天冬酶同源物作为探针，从血细胞文库中鉴定出了一条黑虎虾（Penaeus monodon）半胱天冬酶cDNA同源物（PmCasp）。PmCasp全长1202bp，开放阅读框为954bp，编码317个氨基酸。推导的蛋白质含有一个在大多数半胱天冬酶中保守的潜在活性位点（QACRG五肽）。它与斑节对虾的半胱天冬酶有83%的同一性，与黑腹果蝇的drICE蛋白有30%的同一性，并且在体外表现出半胱天冬酶-3活性。PmCasp在大肠杆菌中克隆并表达，制备了兔多克隆抗血清。在蛋白质免疫印迹中，该抗血清与纯化的重组PmCasp以及含有表达质粒的大肠杆菌裂解物发生反应。在正常虾的粗蛋白提取物中，该抗血清分别与可能对应于无活性的前半胱天冬酶及其蛋白水解中间形式的36kDa和26kDa条带发生反应。通过半定量RT-PCR、蛋白质免疫印迹分析和免疫组织化学法，测定了正常虾以及注射后24小时和48小时感染白斑综合征病毒（WSSV）的虾中PmCasp的表达。半定量RT-PCR分析显示，注射后48小时PmCasp上调，直至濒死状态表达仍保持高水平。蛋白质免疫印迹分析表明，与正常对照虾相比，注射后24小时和48小时PmCasp蛋白水平增加，支持了这些结果。对感染WSSV的虾鳃进行免疫组织化学分析，发现免疫反应性定位于正常细胞和明显凋亡细胞的细胞质中。总之，一种类似半胱天冬酶-3的基因在斑节对虾中保守，并且在WSSV感染后上调。
