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目的克隆与分析日本血吸虫组织蛋白酶L样(SjCLs)基因全长cDNA序列,为进一步研究其功能奠定基础。方法提取日本血吸虫成虫总RNA,经RT-PCR扩增获得cDNA序列;将目的片段连接至pMD18-T Simple载体上,连接产物转化大肠埃希菌,通过酶切和测序鉴定重组质粒;利用生物信息学软件分析序列的开放阅读框、蛋白序列的同源率和功能区。结果以成虫总RNA为模板进行RT-PCR,得到一段长为996 bp的cDNA片段,该片段含有完整读码框,该阅读框编码的蛋白含有331个氨基酸残基,分子量为38.3 kDa,命名为SjCLs;序列比对分析发现该蛋白氨基酸序列与SjCL2同源率最高,达到89.1%;进化树分析发现SjCLs与SjCL2亲缘关系也最近;SjCLs具有类似于SjCL2的N端信号肽,可能经加工后分泌到细胞外;两者所预测的功能基序及三维构象也基本相同。结论 SjCLs具有组织蛋白酶L的基本特征,从而证明研究获得组织蛋白酶L基因家族的一个新成员。
Objective To clone and analyze the full-length cDNA sequence of Schistosoma japonicum cathepsin L (SjCLs) gene and lay a foundation for further study of its function. Methods The total RNA of adult Schistosoma japonicum was extracted and the cDNA sequence was amplified by RT-PCR. The target fragment was ligated into pMD18-T Simple vector and transformed into Escherichia coli. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. The software analyzes the open reading frame of the sequence, the homology of the protein sequence, and the functional region. Results A total length of 996 bp cDNA fragment was obtained by RT-PCR using total RNA of adult samples. The fragment contained a complete reading frame (ORF) encoding 331 amino acid residues with a molecular mass of 38.3 kDa and named SjCLs. The amino acid sequence of this protein shared the highest homology with SjCL2 (89.1%). Phylogenetic tree analysis showed that SjCLs had the closest genetic relationship with SjCL2. SjCLs had N-terminal signal peptide similar to SjCL2, which may be secreted after processing To the extracellular; the predicted functional motifs and three-dimensional conformation are basically the same. Conclusion SjCLs have the basic characteristics of cathepsin L, which proves that the study obtained a new member of cathepsin L gene family.