Expression and characterization of serine proteinase inhibitor of the black tiger shrimp Penaeus monodon

Abstract

Expressed Sequence Tags (ESTs) analysis of the normal and Vibrio harveyi infected haemocyte cDNA libraries of Penaeus monodon identified 6 putative serine proteinase inhibitors that are homologous to a Kazal-type serine proteinase inhibitor (SPI) from crayfish. Four full-length cDNA clones of SPI were obtained. They have the difference in either number of Kazal domain or domain completeness. In this study, we selected a five Kazal-domain clone, sh415, which have 801 bp of open reading frame coding for 266 amino acids. This clone was expressed by using E. coli expression system. NH2-terminal truncated SPI gene, mature protein, was cloned into pTrcHis 2C. A 35 kDa protein band observed in recombinant pTrcHis 2C/SPI clone has higher intensity than that of parental pTrcHis 2C clone. The crude proteins of the recombinant clone were tested for serine proteinase inhibitory activity. In SPI activity gelatin/SDS-PAGE assay, the nondegraded-gelatin band with size of 35 kDa was observed in recombinant clone but not parental clone in trypsin, chymotrypsin, and subtilisin incubation. In inhibitory spectrum assay, trypsin, chymotrypsin, and subtilisin were inhibited their activities to 89 %, 70 %, and 8 %, respectively. The decrease in activity was not observed in elastase.Moreover, we also determined the change in SPI transcripts in haemocytes of V. harveyi challenged P. monodon by using in situ hybridization. We found that Kazal inhibitor expressed in the haemocytes. We discovered that average total haemocyte number was decreased at 6 h after V. harveyi injection. Weak hybridization signal was observed in haemocytes at 6 h after injection indicates the decrease in SPI expression at this time point comparing to others. The number of haemocytes expressing SPI significantly increased at 24 h after injection. Whereas the SPI expressed haemocyte numbers of the others were not significantly different. These results provide preliminary data for further studying SPI.