猪带绦虫45W基因家族cDNA克隆和序列分析

Cloning and Sequencing of Complementary DNA of 45 WGene Family from Taenia solium

根据猪带绦虫45W基因序列设计引物,用反转录-聚合酶链式反应(RT-PCR)对六钩蚴总RNA进行cDNA扩增,扩增产物经琼脂糖凝胶电泳分离、纯化、回收,与载体pGEMT-easyVector连接,转化大肠杆菌JM109,筛选阳性克隆,测序。结果显示,已成功克隆到重要保护性抗原45W基因B型转录本cDNA,完整开放阅读框(ORF)的大小为459bp。序列同源性分析与比较表明,所获得的9个B型转录本cDNA除TSO45W-4B-1和TSO45W-4B-2与已报道的TSO45W-4B同源外,其余均属于首次报道的45W基因家族新成员。各cDNA克隆之间核苷酸序列的差异性为1.5%～19.8%,氨基酸序列之间的差异性为2.6%～29.7%,后者比前者的差异程度更大。

Complementary DNAs (cDNAs) of 45W gene family were amplified from total RNA of hatched and activatedTaenia solium oncosphere using reverse transcription polymerase chain reaction (RT-PCR). The agarose gel electrophoresisof the PCR products showed that the target DNA fragments were specifically amplified. The fragments (492bp) werepurified using DNA purification kit, inserted into pGEM-T Easy Vector, and transformed into JM 109 and then sequenced.The relative sequences were retrieved from GenBank and aligned with our data by DNAstar software. The results showedthat the cDNAs of 9 B-type transcripts of T. solium have been obtained with a 459 bp open reading frame. These transcriptsbelonged to the T. solium 45W gene family with 1.5%-19.8% nucleotide divergence and 2.5%-29.7% amino acid residuedivergence. 7 of 9 transcripts were the new members reported at the first time, while the others almost had the samesequences as TSO45W-4B reported in GenBank.