Cloning, purification and Immunity of α2-Macroglobulin in Freshwater Giant Prawn, Macrobrachium rosenbergii

Autor: Ping Yueh Ho, 何秉岳
Rok vydání: 2009
Druh dokumentu: 學位論文 ; thesis
Popis: 97
The α2-macroglobulin of freshwater giant prawn Macrobrachium rosenbergii was investigated including molecular cloning, expression, protein purification, and immune interactions. The full-length cDNA of the α2-macroglobulin gene was cloned from haemocytes of prawn by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The 4875-bp cDNA contains an open reading frame (ORF) of 4419 bp, encodes a protein of 1472 amino acids with a 23-residue signal sequence. Sequence comparison showed that prawn α2-macroglobulin sequence has overall identity to those of known crustaceans. The α2-macroglobulin was mainly expressed in the prawn haemocytes. Quantitative real-time RT-PCR analysis showed that α2-macroglobulin mRNA transcripts significantly increased to the highest level at C stage of molting. The prawn haemocytes express significant high level of α2-macroglobulin after injection with heat-killed Lactococcus garvieae and Vibrio alginolyticus at 24 h and 12 h, respectively. The copper sulfate challenge reduced the α2-macroglobulin mRNA following exposure for 96 hrs. Prawn α2-macroglobulin was purified from cell-free haemolymph, and the isolation procedure includes DEAE cellulose, Q sepharose fast flow, phenyl-sepharose hydrophobic interaction chromatography, and molecular weight cut-off ultrafiltration. It gives 189-fold purification of prawn α2-macroglobulin with a 2.43% yield. The SDS-PAGE indicates a subunit of 180 kDa with reducing condition. Native α2-macroglobulin weight determined by Ferguson plot and gel filtration was nearly about 430 kDa, suggesting a dimeric configuration with considerable glyco- or lipo- contents. Methylamine inactivation of prawn α2-macroglobulin revealed little mobility changes in native PAGE. It also showed inhibiting effects on PO and SOD activities following co-incubation with haemocytes. In conclusion, this study confirmed that the α2-macroglobulin is highly involved in crustacean immunity.
Databáze: Networked Digital Library of Theses & Dissertations