Expression and characterization of a serine proteinase inhibitors PmSerpin8 from the black tiger shrimp Penaeus monodon / Suphaphon Somnuk = การแสดงออกและลักษณะสมบัติของตัวยับยั้งซีรีนโปรติเนส PmSerpin8 จากกุ้งกุลาดำ Penaeus monodon
SERPIN is a serine proteinase inhibitor found ubiquitously in various organisms including the black tiger shrimp Penaeus monodon. Research studies in Drosophila melanogaster and Manduca sexta revealed that the SERPINs inhibit microbial proteinases and control the proteinase cascades in prophenol oxidase system and blood coagulation. In this study, the PmSERPIN8 from the black tiger shrimp was investigated. The structure of PmSERPIN8 genomic gene was found to have 5 exons and 4 introns. The PmSERPIN8 gene was expressed in shrimp at all developmental stages but highest in nauplii. The PmSERPIN8 gene was expressed mainly in the hemocyte and lesser in gill, lymphoid organ, stomach and epipodite. The PmSERPIN8 expression is up-regulated at 24 and 48 h after V. harveyi and YHV injection, respectively. Using technique in immunocytochemistry, the PmSERPIN8 protein was found in hyaline, semi-granular and granular cells. The expression of PmSERPIN8 protein in the hemocytes was up regulated at 48 h after V. harveyi infection. An open reading frame of PmSERPIN8 gene of 1,254 bp encoding a mature protein of 417 amino acid residues was cloned into the pVR600 expression vector and expressed the rPmSERPIN8 in Escherichia coli BL21(DE3). The recombinant protein (rPmSERPIN8) whose size approximately 43 kDa was expressed as inclusion bodies. The purified rPmSERPIN8 exhibited inhibitory activity against subtilisin and α-chymotrypsin, and the inhibitory activity against subtilisin is not enhanced by heparin. The rPmSERPIN8 was able to inhibit the growth of Bacillus sublilis but not Vibrio harveyi 639. The rPmSERPIN8 was unable to prevent blood coagulation but could impede the proPO activating system. It was most likely that the PmSERPIN8 functioned to control the proPO activating system.