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含有clip结构域的丝氨酸蛋白酶是一类新的丝氨酸蛋白酶家族,可能参与非特异性免疫防御功能。从缢蛏(Sinonovacula constricta)cDNA文库中筛选出一条丝氨酸蛋白酶同源EST序列,然后通过5’RACE扩增、测序,拼接得到全长为1 228 bp的cDNA序列,包括66 bp的5’非翻译区和160 bp的3’非翻译区,以及1 002 bp的开放阅读框。阅读框共编码333个氨基酸,含有17个氨基酸的信号肽序列,并含有发卡结构域(clip domain)和胰蛋白酶样丝氨酸蛋白酶结构域(Tryp_SPc domain)。在clip结构域中包含6个半胱氨酸残基形成的3个二硫键,在Tryp_SPc结构域中包含His-Asp-Ser催化三联体(HDS)。该缢蛏丝氨酸蛋白酶基因被命名为ScSP。实时荧光定量PCR(qRT-PCR)分析表明,ScSP在缢蛏的外套膜、水管、鳃、斧足、性腺、肝胰腺6个组织中均有表达,尤其在肝胰腺中表达显著高于其他组织,其次为性腺组织,而水管,外套膜和鳃中表达量最低。缢蛏经鳗弧菌(Vibrio anguillarum)诱导感染后4 h和8 h,肝胰腺中的ScSP基因表达量显著上调。缢蛏丝氨酸蛋白酶的序列特征与表达分析揭示了ScSP是含有clip结构域的丝氨酸蛋白酶基因,参与了非特异性免疫防御,为进一步研究该基因的结构和功能奠定了基础。
The serine protease containing the clip domain is a novel family of serine proteases that may be involved in nonspecific immune defenses. A homologous EST sequence of serine protease was screened from the cDNA library of Sinonovacula constricta. The full-length cDNA sequence of 1 228 bp was obtained by amplification, sequencing and splicing by 5 ’RACE, including a 66 bp 5’ untranslated region And 160 bp 3 ’untranslated region, as well as a 1 002 bp open reading frame. The reading frame encodes a 333 amino acid, 17 amino acid signal peptide sequence and contains a trypsin-like serine protease domain (Tryp_SPc domain). Three disulfide bridges are formed in the clip domain by six cysteine residues and the His-Asp-Ser catalytic triad (HDS) in the Tryp_SPc domain. The 缢 蛏 serine protease gene was named ScSP. Real-time quantitative PCR (qRT-PCR) analysis showed that ScSP was expressed in 6 tissues of mantle, water tube, gill, axillary, gonad and hepatopancreas in the gall, especially in hepatopancreas, Followed by gonadal tissues, while the lowest expression was found in water mains, mantle and gills. After 4 and 8 h of infection with Vibrio anguillarum, the expression of ScSP gene in hepatopancreas was significantly up-regulated. The sequence characteristics and expression analysis of the serine protease revealed that ScSP is a serine protease gene containing the clip domain, which is involved in nonspecific immune defense and lays the foundation for further study on the structure and function of the gene.