旋毛虫半胱氨酸蛋白酶基因TsCP1的克隆与序列分析

为了研究旋毛虫半胱氨酸蛋白酶的功能,根据GenBank中旋毛虫EST数据库的半胱氨酸蛋白酶基因部分cDNA序列设计引物,以旋毛虫肌幼虫总RNA为模板,进行RT-PCR,克隆到旋毛虫半胱氨酸蛋白酶基因(TsCP1)的完整开放阅读框序列,利用生物信息学软件对其进行预测分析。结果表明,TsCP1cD-NA序列含有一个由1 101个核苷酸组成的开放阅读框,编码由366个氨基酸残基组成的多肽,蛋白质的分子质量理论值为41.9ku,理论等电点为7.46。TsCP1二级结构中α螺旋占31.4%,β折叠占15.3%。TsCP1第1～19位氨基酸残基为信号肽序列,具有3处N-糖基化位点,而且具有半胱氨酸蛋白酶的保守活性位点Cys173,His309及Asn333残基,另外在酶前体序列中有与组织蛋白酶F特征基序ERFNAQ相似的LKFNAQ序列,表明该蛋白属于半胱氨酸蛋白酶家族的组织蛋白酶F亚类。同源性分析表明与其他寄生性蠕虫组织蛋白酶F的一致性在40%以上,系统进化分析表明与吸虫的组织蛋白酶F属于不同的进化分支。

Cloning and sequence analysis of cysteine protease gene TsCP1 of Trichinella spiralis

In order to study the function of cysteine protease of Trichinella spiralis,the open reading frame(ORF) cDNA sequence of cysteine protease of T.spiralis(TsCP1) was cloned by RT-PCR from total RNA of muscle larvae with primers derived from T.spiralis EST sequences in the GenBank database.TsCP1 cDNA sequence was analyzed by bioinformatics software.The results indicated that the TsCP1 cDNA sequence contained an ORF of 1 101 nucleotides and the deduced protein consisted of 366 amino acids with the theoretical molecular weight of 41.9 ku and isoelectric point of 7.46.Analysis of secondary structure revealed 31.4% and 15.3% of α-helix and β-strands,respectively.The signal peptide sequence of TsCP1 located between amino acids 1 and 19 and 3 N-glycosylation sites were identified.The conserved active sites of cysteine protease including Cys173,His309 and Asn333 were identified and the cathepsin F specific motif ERFNAQ like LKFNAQ sequence was revealed in the propeptide of TsCP1,indicating that the TsCP1 belonged to the cathepsin F subgroup of cysteine protease family.TsCP1 protein sequence showed more than 40% identity with other cathepsin F from parasitic helminth,phylogenetic analysis indicated that TsCP1 located in the different clade with trematode cathepsin F.