Purification and molecular cloning of Vibrio protease inhibitor, alpha-2-macroglobulin, from Epinephelus spp.

• Main Authors: Wen-Hsiao Chuang, 莊文?
• Other Authors: Kuo-Kau Lee
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/77534240123350048759

博士 === 國立臺灣海洋大學 === 水產養殖學系 === 97 === Alpha-2-macroglobulin (α-2-M) is a protease inhibitor broadly present in the plasma of vertebrates and invertebrates, and is an important non-specific humoral factor in defence system of the animals. This thesis conducted the purification and characterization of the protease inhibitor, α-2-M, from grouper Epinephelus coioides. Inhibitory activities of purified α-2-M against different proteases secreted by pathogenic bacteria were performed. Rabbit antiserum to the purified α-2-M was used in different immunological methods to determine the variations of the α-2-M among different groupers and fishes. Molecular cloning and characterization of the Thiol ester domain and partial genes of Bait region of α-2-M were compared among different fishes and animal species. The α-2-M of the grouper E. coioides was purified by Fast Protein Liquid Chromatography with various columns including Blue Sepharose 6 Fast Flow, DEAE Sephacel, Con A Sepharose 4B and Phenyl Sepharose High Performance. The purified protein electrophoresed as a single protein band in both native PAGE and non-reduced SDS-PAGE (180 kDa) while electrophoresed as two protein bands (97 and 80 kDa) in reduced SDS-PAGE. In addition, the purified protein was a glycoprotein as positively stained by a glycoprotein staining kit in both non-reduced and reduced SDS-PAGE. Determination the mass of 97 kDa protein contains about 12.4% N-linked glycogen and 80 kDa protein is about 15% by PNGase F enzyme and chemical deglycosylation. As measured by trypsin- N-benzoyl -DL-arginine-p-nitroanilide (BAPNA) assay, the protease inhibitory activities of grouper plasma and the purified protein decreased to 27 and 17%, respectively, at 60℃, and increased at pH 7.0 to 11.0, and decreased when concentration of methylamine increased.The plasma and purified α-2-M showed the high inhibitory activities when the concentration of extracellular products of pathogenic bacteria were between 60 to 200 μg protein/ml, and decreased when concentration of ECP increased. Plasma of Epinephelus bruneus, E. fuscoguttatus, E. lanceolatus, and E. quoyanus exhibited high protease inhibitory activities by BAPNA-trypsin assay. Only one precipitation arc was visualized in each plasma of Epinephelus spp. using the rabbit antiserum to the purified α-2-M of E. coioides, on crossed immunoelectrophoresis (CIE) gels. The plasma of Epinephelus spp. and seawater fish species showed the stronger response than freshwater fish species while that of other animal species showed no response by dot-blot assay. One single band was detected on Native PAGE-Western blotting assay, one single 180 kDa band was detected on non-reduced SDS-PAGE-Western blotting, and four bands (80、97、160、250 kDa) were detected on reduced SDS-PAGE when various grouper plasma was performed. However, no band was detected using plasma from the freshwater fish species and other animal species. Thus further indicates that the protein structure of α-2-M of Epinephelus spp. was closely related among seawater fish species. Identification of the 97 kDa protein significant hit was similar to the α-2-M of Ctenopharyngodon idella, and 80, 160, 250 kDa proteins significant hit were similar to that of Lepomis macrochirus by LC/MS/MS determination. We have isolated 1045 bp partial genes of α-2-M from Epinephelus spp. liver. All sequences contained Thiol ester domain (GCGEQ), two N-linked glycosylation sites, and the 1045 bp genes were deduced and converted into to 347 amino acids. In addition, both of the nucleotide sequences and the deduced amino acid sequences showed high similarity ranging from 94-99% and 96-99%, respectively, among to grouper species. The Thiol ester domain amino acid sequence of grouper α-2-M was further compared with different fish species and known sequences in NCBI GenBank by BioEdit version 7. The unweighted pair group method using arithmetic average (UPGMA) and Neighbor-joining (NJ) were employed to construct the phylogenetic trees of α-2-M among different animal species. The results clustered all species to three major groups (Mammals, Fish species, Crustacean and shellfish) and two minor groups (amphibian and cyclostome). Two different Bait region genes of α-2-M were cloned from E. coioides. High similarity in the end of amino acid sequences in Bait region was determined among different animal species. The amino acid sequence of Bait region in α-2-M after comparing with that of different animal species and reference species, were clustered to Mammals, Fish species, Crustacean and other species. Finally, α-2-M could be detected in groupers and different fish species tested by using immunological assay and cloning of functional domain sequences. The α-2-M exhibited adjuvant effect, therefore, the protease-α-2-M complex could be used as a vaccine candidate to protect fish against microbial infection when protease was an important virulence factor. The α-2-M may have the potential application for fish vaccine development in the future.