关键词: 华北大黑鳃金龟, cDNA表达文库, 丝氨酸蛋白酶, 序列分析, 体外表达

Abstract: Serine proteases are one group of important digestive enzymes in insects. To clarify the characteristics and functions of serine proteases, we used the polyclonal antiserum of peritrophic membrane protein from Trichoplusia ni to screen cDNA expression library of the midgut of Holotrichia oblita, and obtained a full-length cDNA clone encoding serine proteases named as HoSP1 (GenBank accession no. FJ573146). The sequence analysis indicated that HoSP1 is 902 bp in length with an opening reading frame of 783 bp encoding 260 amino acid residues with the predicted molecular weight 26.7 kDa and pI 4.19. Without N-linked glycosylation site, HoSP1 has an O-linked glycosylation site at Thr₁₅₇ and six conservative cysteines forming three pairs of disulfide bonds, which play an important role in sustaining the protein tertiary structure. Amino acid sequence alignment with several kinds of serine proteases showed that HoSP1 has the catalytic active centers of histidine, aspartic acid and serine, and shares significant similarity to 14 kinds of serine proteases from Costelytra zealandica, with the highest identity (52.47%) to CzSP3. After the gene was recombined into pET21b and expressed in vitro, the activity of HoSP1 was determined with BTEE as the substrate, which was 0.0378 μmol/mg·min. The molecular cloning and expression in vitro of HoSP1 lay a foundation for further research of its expression and function in H. oblita.

Key words: Holotrichia oblita, cDNA expression library, serine protease, sequence analysis, in vitro expression