Cloning, mRNA expression, and recombinant expression of peptidoglycan recognition protein II gene from large yellow croaker (Pseudosciaena crocea)

Abstract

Peptidoglycan recognition protein II (pglyrp2) is a type of pattern recognition receptor (PRR) that has amidase activity and is structurally conserved through evolution. However, their contributions in immune defense are different between mammal pglyrp2 and its counterpart in insects. Hitherto, fish pglyrp2 was poorly known in its structure, expression pattern and its contribution in immune defense. In present study, the pglyrp2 gene of the large yellow croaker (Pseudosciaena crocea) was cloned by RACE approach; the full-length cDNA (1842 bp) of pglyrp2 of P. crocea contains a 1446 bp open reading frame that encodes a putative protein of 482 amino acids (aa) with one 21-residue signal peptide. The pglyrp2 fusion protein and mature peptide fusion protein of P. crocea expressed by pET28a vector in the insoluble inclusion bodies (IBs) of Escherichia coli BL21 were confirmed by SDS-PAGE and subsequently purified to homogeneity by Ni-NTA agarose affinity chromatography. In addition, quantitative Real-time PCR (QRT-PCR) assays indicated that large yellow croaker pglyrp2 could be strongly expressed in liver and weakly in gonad, intestine, and stomach. Also, maternally derived pglyrp2 mRNA displayed a high level in unfertilized eggs and low expression throughout embryogenesis and yolk-sac larvae stage. Moreover, as shown in an artificial infection model, the pglyrp2 of P. crocea was confirmed to be a constitutive and inducible acute-phase protein, inducibility of which correlated with activation of anti-oxidant defense response. Thus, pglyrp2 of P. crocea was believed to play an important role in defending the eggs, bacteria recognition and activation of downstream host immune defense.