细粒棘球绦虫未知功能基因384p02i04的克隆及生物信息学分析

摘要

目的：克隆细粒棘球绦虫与药物应激相关的未知功能基因384p02i04序列，并进行生物信息学分析。方法提取细粒棘球绦虫原头蚴总 RNA，采用实时荧光定量聚合酶链反应（RT-PCR）和 cDNA 末端快速扩增技术（RACE）法扩增384p02i04基因片段，测序，并进行生物信息学分析。采用定量反转录聚合酶连锁反应（qRT-PCR）法分析青蒿素、阿苯达唑和吡喹酮等药物干预对384p02i04基因表达的影响。结果384p02i04基因全长449 bp，通过各种生物数据库对该基因进行预测，发现384p02i04基因存在1个编码109个氨基酸的蛋白质开放阅读框，利用 BLAST 数据库将其核酸及氨基酸序列进行比对，并未发现相似度较高的基因及氨基酸，利用 RNA structure软件发现384p02i04基因具有非常低的自由能，其二级结构非常稳定。在青蒿素、阿苯达唑和吡喹酮等药物干预后384p02i04基因均明显下调（P ＜0．05）。结论克隆获得细粒棘球绦虫的一种与药物应激反应相关的未知功能基因，为研发以该基因为靶点的抗包虫病药物奠定基础。%Objective To do the clone and bioinformatics analysis of a novel drug stress-related gene in Echinococcus granulosus .Methods The gene sequence of 384p02i04 was cloned by RT-PCR and RACE methods from protoscolex of E .Granulosus and analyzed by bioinformatics.The gene expressions of 384p02i04 in the protoscoleces of E .Granulosus treated by artemisinin,albendazole and praziquantel were detected by qPCR.Results The size of 384p02i04 gene was 449 bp and there was a protein with open read-ing frame encoded by 109 amino acids.The comparison with the data in BLAST database showed that the gene had low similarity to the known gene.The results in RNA structure analysis showed that the free en-ergy of the gene was very low,and its secondary structure was very stable.Artemisinin,albendazole and praziquantel could down-regulate the gene expressions of 384p02i04 significantly (P <0.001).Conclusion A novel drug stress-related gene 384p02i04 in E .Granulosus was cloned successfully and laid a foundation for the development of anti-echinococcosis drug.