Serpin-14 negatively regulates prophenoloxidase activation and expression of antimicrobial peptides in Chinese oak silkworm Antheraea pernyi

Genes encoding proteins of serpins superfamily are widely distributed in invertebrates. In insects, serpins play important roles in regulating immune responses and other physiological processes. Here, we report the cloning and characterization of cDNA of Apserpin-14 from Chinese oak silkworm (Antheraea pernyi). The Apserpin-14 gene contains 1206 bp open reading frame, encoding a predicted 401 amino acid residue protein. We expressed the recombinant Apserpin-14 protein in Escherichia coli and then purified protein was used to prepare rabbit anti-Apserpin-14 polyclonal antibodies. Quantitative real-time PCR analysis revealed that mRNA level of Apserpin-14 was highest in the fat body, whereas, among developmental stages the 5th instar and pupal stage showed greatest expression. Furthermore, Escherichia coli, Beauveria bassiana, Micrococcus luteus and nuclear polyhedrosis virus challenge enhanced Apserpin-14 transcript in both the fat body and hemocyte. Recombinant Apserpin-14 added to hemolymph inhibited spontaneous melanization and suppressed prophenoloxidase activation stimulated by M. luteus, but did not affect phenoloxidase (PO) activity. Injection of recombinant Apserpin-14 protein into A. pernyi larvae significantly reduced the transcript levels of antimicrobial peptides in the fat body, while its depletion by double stranded RNA enhanced their expression. We concluded that Apserpin-14 likely involved in regulation of proPO activation and production of antimicrobial peptides, implying its important role in the innate immune system of A. pernyi.