有齿食道口线虫ITS及5.8S DNA片段的PCR扩增、克隆及序列分析

PCR Amplification, Cloning and Sequence Analysis of the ITS and 5.8S rDNA of Oesophagostomum Isolates

运用 PCR 方法以保守引物 NC5 及 NC2 扩增了从广东阳江地区猪体分离的食道口线虫 rDNA 的内转录间隔区(ITS)及 5.8S 序列。将 PCR 扩增出的片段纯化后克隆至 pGEM-T Easy 载体,用 PCR 技术及酶切鉴定阳性菌落,对阳性菌落质粒 DNA 进行测序。结果表明,扩增的片段大小为 828 bp,包含部分的 18S、28S 及全部的 ITS-1(362 bp)、5.8S(153 bp)及 ITS-2(217 bp)序列。序列比较表明,该食道口线虫为有齿食道口线虫。本研究在国际上首次报道了中国猪有齿食道口线虫的 ITS 及 5.8S 序列,为食道口线虫的分子生物学的进一步研究奠定了基础。

The internal transcribed spacer (ITS) and 5.8S rDNA of Oesophagostomum spp isolated from Guangdong Province was amplified by PCR using a pair of conserved primers and the amplicons were cloned into pGEM-T Easy vector. The inserts were successfully sequenced, and the results revealed that the inserts were 828 bp in length and consisted of partial 18S, 28S, and complete ITS-1, 5.8S and ITS-2 DNA sequences. The Oesophagostomum isolates were identified as O.dentatum based on ITS-2 sequence. It was the first time that the complete sequence of ITS-1 and 5.8S rDNA of Oesophagostomum dentatum was reported. The results of the present study have laid a foundation for further studies of Oesophagostomum.