Expression and Biochemical characterization of a phospholipase A2 from Photobacterium damselae subsp. piscicida

• Main Authors: Hsu, Po-Yuan, 許博淵
• Other Authors: Lee, Kuo-Kau
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/92505421577853094242

博士 === 國立臺灣海洋大學 === 水產養殖學系 === 102 === This thesis studied the expression of phospholipase gene of Photobacterium damselae subsp. piscicida (Phdp strain 9205) in prokaryotic system, the utilization of gene recombination to produce phospholipase, and the possibility of producing phospholipase in the simple prokaryotic system to replace the comparatively miscellaneous purification step which employed in the past. The extracellular products (ECP) of the Phdp were purified by using various columns (Q Sepharose High Performance and RESOURCE Q) on fast protein liquid chromatography (FPLC) system. Purification of protein was obtained from cut gels and identified as a phospholipase from mass spectroscopy and N-terminal sequence and had a high degree of similarity with GenBank accession no. AB071137 (Naka et al., 2007). An N-terminal sequence was found to be identical with that of another Phdp phospholipase (GenBank accession no. BAB85814) at positions 21 to 30. Using PCR to fetch an 1218 bp fragment from Phdp, and then inserted tis fragment in a pGEMT-easy vector and pET-25b (+) vector to construct an expression system. The constructed bacterial strain induced by the final concentration of 1 mM IPTG. A signal 43 kDa band was determined by sodium dodecyl sulfate-polyacrylamine gel electrophoresis (SDS-PAGE). As the protein and N-terminal sequenced, and a 1-20 amino acid fragment was not induced and was suggested to be a singal peptide. After deletion of 20 amino acid (2.2 kDa), the size of the predicted 45 kDa was almost the same as size 43 kDa. The extracellular products of the Phdp and recombinant protein were visualized as a signal band (43 kDa) after using rabbit antiserum against the recombinant protein in western blotting. The recombination protein was therefore determined to be cloned from Phdp strain 9205. Phosphatidylcholine was degraded by this protein to lysophos- phatidylcholine and a fatty acid. These products were characterized by thin-layer (TLC) and gas chromatography (GC), respectively, allowing the identification of the protein as a phospholipase A2. The recombinant protein had maximum enzymatic activity between pH 4 and 7, and at 40 °C. The activity was inhibited by Zn2+ and Cu2+, activated by Ca2+ and Mg2+, and completely inactivated by dexamethasone and p-bromophenacyl bromide. In the hemolytic activity test, the activity of recombinant protein for cobia was the highest, the second was black seabream, and no haemolytic activity for sheep erythrocytes. A rabbit antiserum against the recombinant protein neutralized the phospholipase A2 activity in the ECP of Phdp strain 9205 and the recombinant protein itself. The recombinant protein was toxic to cobia of about 5 g weight with an LD50 value between 2 and 4 μg protein/g fish. The results revealed phospholipase A2 as a fish toxin in the ECP of Phdp strain 9205. Using high concentration (5 and 10 μg) of recombinant protein to challenge cobia pectoral cell line at 25 ℃ for 24 hr, apparent large number of vacuoles and cell membrane rupture phenomena were observed.