Molecular cloning and characterisation of a proteinase inhibitor, alpha 2-macroglobulin (α2-M) from the haemocytes of tiger shrimpPenaeus monodon

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Abstract

An alpha 2-macroglobulin (α2-M) gene was cloned from the haemocytes of tiger shrimp Penaeus monodon by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the α2-M cDNA consists of 4876 bp with an open reading frame (ORF) of 4494 bp, a 52 bp 5′-untranslated region, and a 327 bp 3′-untranslated region containing a poly A signal. The open reading frame encodes a protein of 1498 amino acids with 18 residues signal sequence. The predicted molecular mass of the mature protein (1480 amino acids) is 167.7 kDa with an estimated pI of 5.30. The P. monodon α2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate α2-Ms. Sequence comparison showed that α2-M deduced amino acid sequence of P. monodon has an overall similarity of 85, 52 and 49% to that of kuruma shrimp Marsupenaeus japonicus, American horseshoe crab Limulus polyphemus and mud crab Scylla serrata, respectively. Alignment of the deduced amino acid sequence to other species α2-M showed that the overall structure is evolutionarily conserved and phylogentic analysis revealed that P. monodon α2-M is closely related to other arthropod α2-M, and displays the highest similarity to M. japonicus α2-M. The α2-M was mainly expressed in haemocytes, but not in eyestalk, gill, muscle, hepatopancreas, and intestine. Quantitative real-time RT-PCR analysis showed that α2-M mRNA transcript in haemocytes of P. monodon increased significantly in 12, 24 and 48 h post-peptidoglycan (PG) injection, but returned to the original values in 72 h post-PG injection.

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