棕蓑猫蛔虫ITS及5.8S rDNA序列的克隆与分析

Cloning and sequence analysis of the ITS and 5.8S rDNA of ascarids from Herpestes urva

以分离自广州动物园棕蓑猫体内的蛔虫为研究对象,用保守引物NC5和NC2对核糖体DNA(rDNA)的内转录间隔区ITS-1、ITS-2及5.8S序列进行PCR扩增,扩增后的片段纯化后克隆至pGEM-TEasy载体,重组质粒通过菌液PCR鉴定后,对其进行序列测定及分析,旨在鉴定棕蓑猫蛔虫的种类。结果显示,目的片段ITS总长为907bp,2个不同样品之间的ITS序列没有差异,与狮弓蛔虫、猫弓首蛔虫和犬弓首蛔虫的ITS-1序列相似性分别为96.9%、71.3%和73.2%;5.8S序列的相似性分别为100%、95.5%和96.8%;ITS-2序列与GenBank中的狮弓蛔虫序列的相似性为94.3%,与猫弓首蛔虫、犬弓首蛔虫序列的相似性均小于60%。结果表明,此次分离的棕蓑猫蛔虫属于狮弓蛔虫。

The internal transcribed spacer（ITS）and 5.8SrDNA of ascarids isolated from Herpestes urvain Guangzhou Zoo were amplified by PCR using apair of conserved primers（NC5and NC2）and the amplicons were cloned into pGEM-T Easy vector respectively.The recombinant plasmids were identified by PCR and the products were sequenced and analyzed.The ITS of the ascarid nematodes from H.urva was 907bp in length and no sequence variation existed between two samples.The sequences similarities were 100%,95.5%and 96.8%respectively for 5.8SrDNA,96.9%,71.3%and 73.2%for ITS-1compared with Toxascaris leonina,Toxocara cati and Toxocara canis available in GenBank,respectively,and 94.3% for ITS-2compared with T.leonina.The ITS-2sequence of the ascarid nematodes from H.urva was significantly different from that of T.cati,T.cansi.It was concluded that the ascarid from H.urvain China may represent T.leonina.