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根据NCBI上登录的溶藻弧菌(Vibrio alginolyticus)全基因组序列,设计一对克隆溶藻弧菌HY9901株Ⅲ型分泌系统“分子尺”蛋白VscP全长基因的特异性引物,PCR扩增获得基因的全长片段。结果显示,vsc P(Gen Bank登录号FR780678)开放阅读框ORF为1 206 bp,共编码401个氨基酸残基。根据推导的氨基酸序列预测其分子质量约为44.4 ku,等电点为5.72。Signal 4.1 Server和TMHMM Server 2.0预测结果表明,VscP无信号肽与跨膜结构域。Soft Berry-Psite预测结果显示,VscP含有3个N端糖基化位点,2个cAMP及c GMP依赖性蛋白激酶磷酸化位点,5个蛋白激酶C磷酸化位点,9个酪蛋白激酶II磷酸化位点,2个N端豆蔻酰基化位点,1个酰胺化位点和3个微体C末端靶信号位点。运用MEGA6.0构建的VscP系统进化树显示,溶藻弧菌VscP与副溶血弧菌(V.parahaemolyticus)聚为一簇。应用SWISS-MODEL软件构建VscP亚基三维结构模型发现,其与鞭毛Fil K蛋白有相似构型。
According to the whole genome sequences of Vibrio alginolyticus registered on NCBI, a pair of specific primers were designed to amplify the full-length gene of type Ⅲ secretion system “molecular ruler ” of V. alginolyticus HY9901, and PCR amplification The full-length fragment of the gene is obtained. The results showed that the open reading frame (ORF) of vsc P (Gen Bank Accession No. FR780678) was 1 206 bp, encoding a total of 401 amino acid residues. According to the deduced amino acid sequence, the predicted molecular mass was about 44.4 ku and the isoelectric point was 5.72. Signal 4.1 Server and TMHMM Server 2.0 predict that VscP has no signal peptide or transmembrane domain. Soft Berry-Psite predicts that VscP contains three N-terminal glycosylation sites, two cAMP and c-GMP dependent protein kinase phosphorylation sites, five protein kinase C phosphorylation sites, nine casein kinase II phosphorylation sites, two N-myristoylation sites, one amidation site and three C-terminal microRNA signaling sites. The VscP phylogenetic tree constructed with MEGA6.0 showed that Vibrio alginolyticus VscP clustered with V. parahaemolyticus. Using the SWISS-MODEL software to construct the 3D structural model of VscP subunit, it was found that it has a similar configuration with flagellum Fil K protein.