Lewis E, Hunter S J, Tetley L, Nunes C P, Bazzicalupo P, Devaney E
Department of Veterinary Parasitology, University of Glasgow, UK.
Mol Biochem Parasitol. 1999 Jun 25;101(1-2):173-83. doi: 10.1016/s0166-6851(99)00070-5.
A fragment of a cut-1 like gene from the filarial nematode Brugia pahangi (designated Bp-cut-1) was isolated by PCR from genomic DNA. The sequence was used to design primers for use in RT-PCR and resulted in the isolation of a cDNA fragment from larvae in the process of the L3-L4 moult. Screening of a B. malayi genomic library identified a single clone, Bm-cut-1. Using primers designed from the Brugia sequences, semi-quantitative RT-PCR was carried out on 11 different life cycle stages chosen to cover periods around the moult and inter-moult periods. This analysis demonstrated that the cut-1 mRNA was most abundant preceding the moult, consistent with its function as a cuticular protein. Immuno-gold electron microscopy using an affinity purified antiserum raised to the highly conserved region of Ascaris CUT-1 confirmed that the protein was restricted to a tight band in the median layer of the cuticle. Despite the fact that no transcripts could be detected in mature adult worms by RT-PCR, immuno-gold microscopy revealed staining of the microfilarial cuticle within the uterus of the adult female worm, suggesting that other cut-1-like genes are present in Brugia.
通过聚合酶链反应(PCR)从丝状线虫马来布鲁线虫(Brugia pahangi)的基因组DNA中分离出一个类cut-1基因片段(命名为Bp-cut-1)。该序列被用于设计逆转录聚合酶链反应(RT-PCR)引物,并在L3-L4蜕皮过程中从幼虫中分离出一个互补DNA(cDNA)片段。对马来布鲁线虫基因组文库的筛选鉴定出一个单一克隆Bm-cut-1。使用根据布鲁线虫序列设计的引物,对选择用于涵盖蜕皮期和蜕皮间期前后时间段的11个不同生命周期阶段进行了半定量RT-PCR。该分析表明,cut-1信使核糖核酸(mRNA)在蜕皮前最为丰富,这与其作为表皮蛋白的功能一致。使用针对蛔虫CUT-1高度保守区域产生的亲和纯化抗血清进行免疫金电子显微镜检查,证实该蛋白局限于表皮中层的一条紧密带中。尽管通过RT-PCR在成熟成虫中未检测到转录本,但免疫金显微镜显示成年雌虫子宫内微丝蚴表皮有染色,这表明布鲁线虫中存在其他类cut-1基因。