重庆荣昌犬钩虫ITS及5.8S rDNA的克隆及序列分析

On Cloning and Sequence Analysis of ITS and 5. 8S rDNA of Ancylostoma Caninum in Rongchang,Chongqing

利用聚合酶链式反应(PCR)扩增犬钩虫rDNA的ITS及5.8S序列,然后将PCR扩增产物回收纯化后连接到pMDTM19-T载体上进行克隆.重组质粒通过菌落PCR鉴定,将阳性重组质粒进行序列测定并且进行序列分析.结果显示,5株犬钩虫荣昌分离株ITS序列的总长为833 bp^834 bp.ITS-1序列总长无差异,但存在个别碱基的差异;5.8S序列总长与序列均无差异;ITS-2序列总长存在差异(221 bp^222 bp).通过与GeneBank上报道的其他钩虫的ITS序列进行相似性分析,发现犬钩虫荣昌分离株(RCF)与犬钩虫广州分离株相似性最高,为99.9%,与美国北海狮弯口属钩口线虫ITS序列的相似性最低,为86.1%.表明ITS可作为分子标记用于犬钩虫种属以及种间的鉴定.该研究结果旨在为今后犬钩虫的分类鉴定、种群遗传关系、分子流行病学调查以及对该病的防治等方面的更深入研究奠定基础.

The Internal transcribed spacer （ITS） and 5.8S sequences of Ancylostoma caninum isolated in Rongchang have been amplified by PCR and the amplicons cloned into pMDTM19-T Vector, respectively. The inserts have successfully been sequenced, and the ITS and 5.8S sequences of the five A. caninum isolates in Rongchang vary 833--834 bp in length. The analysis of sequence reveals that the ITS-1 sequences＇ total length is almost the same except for several bases and the 5.8S sequences are totally all the same. These ITS-2 sequences are 221--222 bp in length. It shows that the identity of ITS of A. caninum RCF isolate in Rongchang is up to 99.9 % with A. caninum isolated in Guangzhou, while it is down to 86.1 % with Uncinaria lucasi. It suggests that ITS can be applied to interspecific identification as a useful molecu- lar marker. The results of this study lay down the foundation for further study on molecular epidemiology, species genetic relationships and diagnostics of A. caninum.