阴道毛滴虫半胱氨酸蛋白酶基因变异分析

Genetic Variation and Clustal Analysis of Trichomonas vaginalis Cysteine Proteases

目的研究阴道毛滴虫半胱氨酸蛋白酶(TvCP)基因变异特点,为分子种系发生和遗传进化等提供参考资料。方法利用PCR扩增TvCP基因,用pBS-T或pET28b载体构建重组质粒,转化大肠埃希菌,筛选阳性克隆,测定目的基因序列。通过以序列相似性为基础的数据库搜索方法检索同源序列,利用Clustal X、DNAstar等软件分析TvCP基因核苷酸和氨基酸序列多态性、遗传变异和进化规律。结果克隆的TvCP2和TvCP3基因与数据库中相应参考序列在核苷酸和氨基酸水平上的相似性均为99%以上,高度保守。克隆的TvCP4基因与TvCP4参考序列在核苷酸和氨基酸水平上的相似性为97.5%以上,属于TvCP4基因家族的两个不同成员。根据阴道毛滴虫基因组数据库信息,经分析表明TvCP4酶前体由305个氨基酸残基组成,共有大小一致的TvCP4同源分子9个和N-末端缺少部分氨基酸残基的同源分子2个,它们之间的氨基酸序列相似性为62.3%～96.7%。TvCP4、TvCP1-3、TvCP12、TvCP25和半胱氨酸蛋白酶65(CP65)之间也有同源性,氨基酸序列相似性约为61%～88.2%。此外,阴道毛滴虫还拥有为数众多的其他组织蛋白酶L样成员,TvCP构成呈高度多样性。序列联配和聚类分析表明TvCP酶活性位点、酶前体结构中ERFNIN样基序和形成二硫键的半胱氨酸残基数目和位置非常保守,进化树分析也表明它们可能由共同的祖先分子通过基因复制和不断突变进化而来。结论TvCP组成一蛋白酶家族,家族内各成员酶活性位点上残基高度保守,但其他位点上的序列呈现高度多态性;推测TvCP家族各成员可能由一个共同的祖先基因分子进化而来,在保持酶基本功能的同时又赋予多样的功能。

Objective To clone the genes coding for cysteine proteases （CPs, TvCPs） from Trichomonas vaginalis and to analyze their genetic variations with the related sequences from NCBI database （GenBank） and T. vaginalis Genome Project database from The Institute for Genomic Research （TIGR）. Method TvCP genes were amplified using PCR, and inserted into vector pET28b or pBS-T. The recombinant plasmids were then transformed to Escherichia coli BL21 or Top10 strain. The recombinant plasmids were used for sequencing. Homologous TvCP genes were blasted based on NCBI GenBank and TIGR T. vaginalis Genome Project database. The sequences of cloned TvCP genes were aligned and clustered by Clustal X （1.83 version） with retrieved sequences. Comparisons of amino acids among cathepsin L-like TvCPs, human L-like cathepsins and papaya papain were performed using DNAstar software, and their phylogenic tree was constructed based on neighbor-joining method using Clustal X. Results Two TvCP3 clones and one TvCP2 had a high identity of more than 99% with their responding TvCPs. Three clones of TvCP4 genes, GZ-CP4-clone 1-3, belonged to two members of a family showing a high percentage identity of more than 97.5% with the sequences of TvCP4 genes from databases （GenBank and TIGR） both at amino acid and nucleotide levels. Nine homologous TvCP4 pro-enzymes with 304 amino acids and other two members with deletions of N-terminal sequence existed in T. vaginalis sharing a similarity of 62.3-96.7% amino acids, which may evolve by means of gene replication and deletion. TvCP1-4, TvCP12, TvCP25 and CP65 had an identity of 61-88.2% at amino acid levels. So far, all reported sequences of C1 family from T. vaginalis belonged to capanthesin L-like subfamily with the same enzymatic active sites, conserved cysteine residues and similar structural features such as ERFNIN-like motif in pro-enzyme region, suggesting that they might result from gene duplication and mutations. Conclusion TvCPs belong to cathepsin L-like family with genetic diversity, but they have the same active amino acid residues, cysteine residues and similar structural characteristics, suggesting that they may derive from one ancestor.