日本血吸虫磷酸丙糖异构酶编码基因的克隆与序列测定

目的克隆和鉴定日本血吸虫磷酸丙糖异构酶(SjTPI)编码基因,为寻找血吸虫病的候选疫苗联合应用打基础.方法设计合成引物,抽提日本血吸虫成虫总RNA,用RT-PCR法从中扩增出SjTPI基因编码序列,将其克隆入pGEM-T载体,用双酶切、以重组质粒为模板进行PCR扩增和测序进行鉴定.结果 RT-PCR法从成虫总RNA中扩增出大小为759 bp SjTPI基因编码序列,重组质粒pGEM-SjTPI经双酶切、PCR扩增,均可获得一条与RT-PCR产物一致的DNA片段,序列测定结果表明具有一个长度为759 bp的完整开放阅读框,与日本血吸虫(菲律宾株)和曼氏血吸虫磷酸丙糖异构酶核苷酸序列有高度同源性(分别为99%和88%).结论该实验成功地克隆了SjTPI编码基因,为进一步研究提供了条件.

Cloning and sequencing of triosephosphate isomerase gene of schistosoma japonicum

Objective To clone the gene encoding triosephosphate isomerase of schistosoma japonicum (SjTPI) for discovering candidate vaccine and diagnostic molecular for schistosomiasis. Methods The specific primers were designed and synthesized. The DNA encoding triosephosphate isomerase of S. japonicum was amplified by polymerase chain reaction using SjcDNA as template. The PCR products was cloned into pGEM-T vector and the inserted DNA fragments were comfirmed by both restriction endonuclease digestion and PCR followed by DNA sequencing. Results For PCR, a specific band of 759 bp was amplified with female SjcDNA. The same band was obtained by double restriction of recombinant plasmids (pGEM-SjTPI) and PCR using recombinant plasmids as template. Of the sequence generated, the nucleotide sequence has an open reading frame of 759bp. Comparison of the sequence with SjTPI gene (Phillipine) and SmTPI revealed considerably high homologies (99 9% and 88%). Conclusion The findings suggest that SjTPI gene is successfully cloned. It provides the basis for further study on SjTPI expression and its immunological prevention.