|ชื่อเรื่อง||:||Inhibitory activity of Kazal-type serine proteinase Inhibitor from the black tiger shrimp Penaeus Monodon and crayfish Pacifastacus Leniusculus|
|คำค้น||:||Serine proteinase inhibitor , Pacifastacus leniusculus|
|ผู้ร่วมงาน||:||Vichien Rimphanitchayakit , Anchalee Tassanakajon , Soderhall, Kenneth , Chulalongkorn University. Faculty of Science|
Thesis (D.Sc.)--Chulalongkorn University, 2009
Serine proteinase inhibitors are found ubiquitously in living organisms and involved in homeostasis of processes using proteinases as well as innate immune defense. Two two-domain Kazal-type serine proteinase inhibitors, KPI2 and KPI8, have been identified from the hemocyte cDNA library of the crayfish Pacifastacus leniusculus. Unlike other KPIs from P. leniusculus, they are found specific to the hemocytes and contain an unusual P2 amino acid residue, Gly. To unveil their inhibitory activities, the two KPIs and their domains were over-expressed. By testing against subtilisin, trypsin, chymotrypsin and elastase, the KPI2 was found to inhibit strongly against subtilisin and weakly against trypsin, while the KPI8 was strongly active against only trypsin. With its P1 Ser and Lys, the KPI2_domain2 and KPI8_domain2 were responsible for strong inhibition against subtilisin and trypsin, respectively. Mutagenesis of KPI8_domain1 at P2 amino acid residue from Gly to Pro, mimicking the P2 residue of KPI8_domain2, rendered the KPI8_domain1 strongly active against trypsin, indicating the important role of P2 residue in inhibitory activities of the Kazal-type serine proteinase inhibitors. Only the KPI2 was found to inhibit against the extracellular serine proteinases from pathogenic fungus of the freshwater crayfish, Aphanomyces astaci The five-domain Kazal-type serine proteinase inhibitor SPIPm2 from the black tiger shrimp Penaeus monodon is presumably involved in innate immune response. The SPIPm2 with the domain P1 residues Thr, Ala, Glu, Lys and Glu was isolated from the hemocyte cDNA libraries and found to strongly inhibit subtilisin and elastase, and weakly inhibit trypsin. To unravel further the inhibitory activity of each domain, we subcloned, over-expressed and purified each individual SPI domain. Their inhibitory specificities against trypsin, subtilisin and elastase were determined. Domain 1 was found to be inactive. Domains 2, 3 and 5 inhibited subtilisin. Domain 2 inhibited also elastase. Domain 4 weakly inhibited subtilisin and trypsin. The intact SPIPm2 inhibitor was found to possess bacteriostatic activity against the Bacillus subtilis but not the Bacillus megaterium, Staphylococcus aureus, Vibrio harveyi 639 and Escherichia coli JM109. Domains 2, 4 and 5 contributed to this bacteriostatic activity. Mutagenesis of the P2′ residue of the domain 1 and domain 3 of SPIPm2 revealed that P2′ position of Kazal-type SPI might involve in binding efficiency against proteinase. To delineate the genomic organization of the SPI gene, the primers specific to SPIPm2 were designed to PCR amplify the genomic version of the gene. The amplified DNAs were sequenced and aligned with the cDNA clone. The results showed that the open reading frame of the gene contained 7 exons and 6 introns. Western blot using anti-SPIPm2 of total proteins from different shrimp tissues revealed that the SPIPm2 was mainly produced in the hemocytes though lower amount was found in gill, heart, epipodite, stomach and lymphoid. In addition, SPIPm2 was found in the plasma. To determine the effect of SPIPm2 on WSSV replication, the shrimp were injected with rSPIPm2 and WSSV. It was found that SPIPm2 had the anti-viral activity against WSSV.
Suchao Donpudsa . (2552). Inhibitory activity of Kazal-type serine proteinase Inhibitor from the black tiger shrimp Penaeus Monodon and crayfish Pacifastacus Leniusculus.
กรุงเทพมหานคร : จุฬาลงกรณ์มหาวิทยาลัย.
Suchao Donpudsa . 2552. "Inhibitory activity of Kazal-type serine proteinase Inhibitor from the black tiger shrimp Penaeus Monodon and crayfish Pacifastacus Leniusculus".
กรุงเทพมหานคร : จุฬาลงกรณ์มหาวิทยาลัย.
Suchao Donpudsa . "Inhibitory activity of Kazal-type serine proteinase Inhibitor from the black tiger shrimp Penaeus Monodon and crayfish Pacifastacus Leniusculus."
กรุงเทพมหานคร : จุฬาลงกรณ์มหาวิทยาลัย, 2552. Print.
Suchao Donpudsa . Inhibitory activity of Kazal-type serine proteinase Inhibitor from the black tiger shrimp Penaeus Monodon and crayfish Pacifastacus Leniusculus. กรุงเทพมหานคร : จุฬาลงกรณ์มหาวิทยาลัย; 2552.