旋毛虫组织蛋白酶B基因TsCB1的克隆与生物信息学分析

Cloning and bioinformatics analysis of cathepsin B gene TsCB1 of Trichinella spiralis

为了研究旋毛虫组织蛋白酶B的功能,对GenBank中旋毛虫基因组数据库和EST数据库进行检索,获得旋毛虫组织蛋白酶B的cDNA序列并设计引物。以旋毛虫肌幼虫总RNA为模板,进行RT-PCR,将PCR产物克隆到pMD18-T载体后测序并进行生物信息学分析。结果表明,成功克隆到旋毛虫组织蛋白酶B基因(TsCB1)的cDNA序列,TsCB1cDNA含有1个由1 449个核苷酸组成的完整的开放阅读框,编码482个氨基酸残基组成的多肽,蛋白质分子质量理论值为55.1ku,理论等电点为7.66。TsCB1第1～35位氨基酸残基为信号肽序列,有2个潜在的N-糖基化位点,具有1个生长调节素B结构域和半胱氨酸蛋白酶结构域,半胱氨酸残基活性位点被丝氨酸残基所替换,同源性分析表明与其他线虫组织蛋白酶B的一致性在60%左右。

In order to study the function of cathepsin B of Trichinella spiralis ,primers derived from T. spiralis genome and EST database in GenBank were designed and the open reading frame（ORF） se- quence of cathepsin B（TsCB1） was cloned by RT-PCR from T. spiralis muscle larvae,and bioinformatics analysis was performed. The results indicated that the TsCB1 cDNA sequence contained an ORF of 1 449 nucleotides and the deduced protein consisted of 482 amino acids with a theoretical molecular weight of 55.1 ku and an isoelectric point of 7.66. The deduced protein had a signal peptide sequence between amino acids 1 and 35,2 N-glycosylation sites,1 somatomedin B-2 domain and 1 cysteine protease domain with the cysteine active site changed to serine. TsCB1 protein sequence showed about 60% identity with cathepsin Bs from other parasitic nematodes.