Journal of Biotechnology 131 (2007) 9–19 Cloning and characterization of a caspase gene from black tiger shrimp (Penaeus monodon)-infected with white spot syndrome virus (WSSV)  Kanokpan Wongprasert a , Pakkakul Sangsuriya b , Amornrat Phongdara c , Saengchan Senapin b,d,∗ a Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand b Center of Excellence for Shrimp Molecular Biology and Biotechnology, Mahidol University, Bangkok 10400, Thailand c Center for Genomic and Bioinformatics Research, Faculty of Science, Prince of Songkla University, Songkhla, 90112, Thailand d National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand Received 12 March 2007; received in revised form 2 May 2007; accepted 25 May 2007 Abstract 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 1202 bp with a 954 bp 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 26 kDa 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 48 h 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 48 h 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 48 h 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. © 2007 Elsevier B.V. All rights reserved. Keywords: Apoptosis; Caspase; Penaeus monodon; Shrimp; WSSV; ICE 1. Introduction Apoptosis or programmed cell death is involved both in embryonic development and in defense against viral pathogens. It is also responsible for inducing cell death from a wide vari- ety of signals such as UV, toxins, hormones, serum growth factor deprivation, chemotherapeutic agents, ionizing radiation and autoimmune disorders (Tomei et al., 1993). The process is evolutionarily conserved across animal taxa. Most of the  The GenBank accession no. for the Penaeus monodon caspase (PmCasp) cDNA sequence reported in this paper is DQ846887. ∗ Corresponding author at: Center of Excellence for Shrimp Molecular Biol- ogy and Biotechnology, Mahidol University, Bangkok 10400, Thailand. Tel.: +66 2 2015889; fax: +66 2 3547344. E-mail address: saengchan@biotec.or.th (S. Senapin). morphological changes resulting from apoptosis are caused by activation of enzymes in the caspase family. Currently, more than a dozen caspase family members have been discovered and classified relative to their involvement in inflammation or pro- grammed cell death (see reviews by Cohen, 1997; Nicholson and Thornberry, 1997; Thornberry and Lazebnik, 1998). Some upstream caspases hierarchically activate others in a proteolytic cascade that creates an amplification circuit. Activation of down- stream executioner caspases (caspase-3, -6, and -7) leads to proteolytic cleavage and concomitant inactivation or activation of their specific cellular protein targets to yield the physiological and morphological changes characteristic of apoptosis (Grutter, 2000). For example, cleavage of nuclear lamin is required for nuclear shrinking and budding, and cleavage of the cytoskeleton proteins fodrin and gelsolin causes the loss of overall cell shape (Hengartner, 2000). 0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.05.032