大熊猫蛔虫ITS及5.8SrDNA序列的克隆与分析

基于核糖体DNA第一与第二转录间隔序列以及5.8S序列,以分离自广州动物园大熊猫体内的蛔虫为研究对象,用保守引物NC_5和NC_2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,对阳性菌落进行序列测定及分析,鉴定大熊猫蛔虫的种类。结果显示,目的片段总长为910 bp,2个不同样品之间的ITS及5.8S序列没有差异,与GenBank~（TM）中的拜林蛔线虫（Baylisascaris transfuga）、猪蛔虫（Ascaris suum）和人蛔虫（Ascaris lumbricoides）的ITS序列相似性分别为96.6%、82.9%和82.7%。结果表明,此次分离的大熊猫蛔线虫可能为拜林蛔线虫。

Cloning and Sequence Analysis of ITS and 5.8S rDNA of Ascarid Nematodes from Giant Panda

To identify the species of ascarid samples isolated from a giant panda in Guangzhou Zoo,China,we designed a pair of conserved primers（NC2/NC5） to amplify the first and second internal transcribed spacers（ITS） and 5.8S of ribosomal DNA（5.8S rDNA）.The ITS rDNA was amplified by PCR from the ascarid samples.The amplicons were then purified and cloned into pGEM - T Easy vector.After being identified by PCR amplification and restriction digestion,the positive clones were sequenced and the sequences were compared with those of Baylisascaris transfuga,Ascaris sum and Ascaris lumbricoides available in GenBank~（TM）.The results revealed that the two ascarid specimens had identical ITS and 5.8S rDNA sequences of 910bp length.The ITS sequences of the ascarid nematodes from the giant panda were significantly different from those of Baylisascaris transfuga,Ascaris sum,and Ascaris lumbricoides,with similarities of 96.6%,82.9%,and 82.7%,respectively. The ascarid nematodes from the giant panda differed from the species listed above and their ITS sequence had not been previously reported.We provisionally identify the species as Baylisascaris transfuga,the species showing greatest similarity.