Molecular cloning and characterization of alpha 2-macroglobulin (alpha2-M) from the haemocytes of Chinese mitten crab Eriocheir sinensis.

ABSTRACT

Alpha 2-macroglobulin (alpha2-M) is a large protein in blood and is able to inactivate a variety of proteinases in invertebrates and vertebrates. The alpha2-M gene was obtained from the Chinese mitten crab, Eriocheir sinensis, by the reverse transcript-polymerase chain reaction (RT-PCR) and the rapid amplification of cDNA ends (RACE). The alpha2-M cDNA of E. sinensis contained 4851 bp with an open reading frame of 4371 bp, a 70 bp 5'-untranslated region, a 410 bp 3'-untranslated region, and three common putative functional domains. The putative domains include a bait region, a GCGEQNM thiol ester domain, and a receptor-binding domain in E. sinensis, which are similar to those in other species. Sequence comparison indicates that the alpha2-M deduced amino acid sequence of E. sinensis has an overall identity of 44%, 44%, 43% and 38% to that of Marsupenaeus japonicus, Macrobrachium rosenbergii, Litopenaeus vannamei and Scylla serrata, respectively. Alignment of deduced amino acid sequence to other species shows that the overall structure of alpha2-M is evolutionarily conserved. Phylogenetic analysis reveals that the E. sinensis alpha2-M is closely related to the alpha2-Ms in other arthropods. Quantitative PCR analysis showed that alpha2-M was mainly expressed in haemocytes, but not in gill, muscle, hepatopancreas, gut and stomach. Its mRNA transcript in haemocytes of E. sinensis increased significantly at 12, 24 and 48 h post-Aeromonas hydrophila (Gram negative bacteria) injection, indicating that A. hydrophila could induce alpha2-M mRNA expression in E. sinensis.