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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902bp,开放阅读框(ORF)长783bp,编码260个氨基酸,推测分子量和pI值分别为26.7kDa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0.0378μmol/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。
In order to understand the molecular characteristics and functions of these enzymes, serine protease was used to screen the cDNA of Holotrichia oblita midgut cDNA from Trichoplusia ni peripheral coat protein polyclonal antibody Expression library, and for the first time, the cDNA sequence encoding the sericin proteinase from Chrysolophus americana was named HoSP1 (GenBank accession number FJ573146). Sequence analysis showed that the gene was 902 bp in length and 783 bp in length with an open reading frame (ORF) encoding 260 amino acids. The deduced molecular weight and pI value were 26.7 kDa and 4.19, respectively, and did not contain N-glycosylation sites. However, An O-glycosylation site, containing six conserved cysteine residues, constituting three pairs of disulfide bonds, plays an important role in maintaining the tertiary structure of the protein. By comparing with several serine proteases, the enzyme has the catalytic activity of histidine (His), aspartic acid (Asp) and serine (Ser), and 14 serine proteases of Costelytra zealandica There was obvious similarity, among which the sequence identity with CzSP3 was the highest, which was 52.47%. After the recombination with pET21b vector, the gene was expressed in vitro. Using BTEE as substrate, the activity of this gene was 0.0378μmol / mg · min. Cloning and in vitro expression of HoSP1 gene provide a basis for further study of the expression and function of this enzyme in Sciurus chinensis.