Cloning and expression analysis of a fungal protease inhibitor gene CP8 in Antheraea pernyi(Lepidoptera: Saturniidae)
Protease inhibitors play important roles in the insect immunity. Here, we isolated a fungal protease inhibitor gene from Antheraea pernyi(Lepidoptera: Saturniidae) for the first time. The gene was named ApCP8. ApCP8 contains 318 nucleotides with a putative open reading frame(ORF) encoding 106 amino acid residues(with a signal peptide of 19 amino acid residues). The isoelectric point(p I) and predicted molecular weight of ApCP8 protein were 9.05 and 11.3 k Da, respectively. Amino acid sequence and phylogenetic analysis among A. pernyi and other insects represented that these fungal protease inhibitors had a high homology, and ApCP8 existed in one evolutionary branching with Antheraea mylitta's. RT-qPCR results showed that ApCP8 displayed the highest expression level in the fat body. In addition, ApCP8 can be upregulated by Beauveria bassiana in 0.5 h after injection, which indicated that it may be involved in the antifungal immune response of Antheraea pernyi. Meanwhile, the recombinant protein of As-ApCP8 was expressed successfully by pET-32a(+) vector, and the antiApCP8 antibody against rabbit was prepared. This research will provide a preliminary basis for further antifungal immune function exploration of ApCP8.