华北大黑鳃金龟中肠丝氨酸蛋白酶cDNA克隆、 序列分析及表达

丝氨酸蛋白酶是昆虫体内一类重要的消化酶， 为了了解该类酶的分子特性及功能， 本研究利用粉纹夜蛾Trichoplusia ni围食膜蛋白多克隆抗体筛选华北大黑鳃金龟Holotrichia oblita中肠cDNA表达文库， 首次得到编码华北大黑鳃金龟丝氨酸蛋白酶cDNA序列， 命名为HoSP1（GenBank登录号为FJ573146）。序列分析表明， 该基因长902 bp， 开放阅读框（ORF）长783 bp， 编码260个氨基酸， 推测分子量和pI值分别为26.7 kDa和4.19， 不含有N-糖基化位点， 但在Thr₁₅₇处有一个O-糖基化位点， 含有6个保守的半胱氨酸残基， 组成3对二硫键， 对于维持蛋白质的三级结构起着重要的作用。通过与几种丝氨酸蛋白酶的比对发现， 该酶具有组氨酸（His）、 天冬氨酸（Asp）、 丝氨酸（Ser）催化中心， 与褐新西兰肋翅鳃金龟Costelytra zealandica的14种丝氨酸蛋白酶有明显的相似性， 其中与CzSP3的序列一致性最高， 为52.47%。把该基因与pET21b载体重组后， 进行体外表达， 以BTEE为底物， 测得该酶的活力为0.0378 μmol/mg·min。HoSP1基因的克隆及体外表达为进一步研究该酶在华北大黑鳃金龟体内的表达及功能提供了依据。

Molecular cloning, sequence analysis and expression of serine protease cDNAs from the midgut of Holotrichia oblita (Coleoptera: Melolonthidae)

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.