Characterization of PmSERPIN3 gene from black tiger shrimp Penaeus monodon

Serpin or serine proteinase inhibitor is a family of a proteinase inhibitor that involves in controlling the proteolytic cascade in various biological processes and has been identified in most of organisms. In shrimp, several serpins have been identified so far. In this study, PmSERPIN3 gene was characterized. From the 5\'- and 3\'- Rapid Amplification of cDNA Ends (RACE) techniques, the full-length of PmSERPIN3 cDNA is about 1,456 bp containing an open reading frame of 1,233 bp encoding for 410 amino acid residues with 23 amino acid residues signal peptide was obtained. Genome sequence analysis revealed that the PmSERPIN3 was an intronless gene. RT-PCR analysis revealed that it expressed in all shrimp tissues tested. Moreover, the expression of PmSERPIN3 was also found in all developmental stages including nauplius, zoea, mysis and adult. The expression level of PmSERPIN3 gene did not respond to Vibrio harveyi, white spot syndrome virus and yellow head virus challenges. Using the immunofluorescent staining observed under confocal laser scanning microscope, the result revealed that PmSERPIN3 appeared in 3 main types of hemocytes such as hyaline, semigranular, and granular hemocytes and was up-regulated upon V. harveyi infection. The recombinant PmSERPIN3 (rPmSERPIN3) was successfully produced in Escherichia coli. The proteinase inhibitory activity assay revealed that the purified rPmSERPIN3 could inhibit subtilisin. Interestingly, rPmSERPIN3 also inhibited the shrimp prophenoloxidase system activation in vitro. The effect of PmSERPIN3 on bacterial clearance of shrimp after V. harveyi infection was tested. The results revealed that the number of total bacteria and V. harveyi (CFU/ml) after V. harveyi and rPmSERPIN3 injection at 30 min was higher than the control shrimp for 3.5 and 2.9 fold, respectively. However, rPmSERPIN3 could not inhibit the clot formation of shrimp hemolymph. Taken together, it can be implied that PmSERPIN3 functions as an inhibitor of proPO system activation. Its inhibitory activity takes part in the bacterial clearance efficacy of shrimp.