日本血吸虫酪氨酸羟化酶基因的克隆和序列分析

目的　克隆并鉴定日本血吸虫酪氨酸羟化酶(TH)基因,探讨其他信号蛋白在信号传导中对TH的调节及其之间的分子作用机制,从而为血吸虫新型疫苗的设计和药物开发开辟新的途径。　方法　以曼氏血吸虫的 TH cDNA为模板设计引物,以日本血吸虫成虫mRNA为模板逆转录合成cDNA链,将扩增出的日本血吸虫TH蛋白编码基因序列,克隆入 pGEM T easy载体,用单酶双切法和DNA测序进行鉴定,并与曼氏血吸虫的TH基因序列进行同源性比较。　结果　自日本血吸虫RNA经逆转录得到一长度为 480 bp的 DNA片段,经测序与曼氏血吸虫 TH的同源性为 87%。结论　成功扩增出日本血吸虫TH的中间编码区域,为进一步扩增日本血吸虫TH基因全长以及后续实验奠定了基础。

CLONING AND SEQUENCE ANALYSIS OF TYROSINE HYDROXYLASE GENE OF SCHITOSOMA JAPONICUM

Objective To clone and identify tyrosine hydroxylase gene of Schistosoma japonicum (SjTH) for approach to the role of SjTH in signal transduction of S. japonicum and for new vaccine molecules against schistosomiasis. Methods A pair of primers were designed and synthesized based on the cDNA sequence of tyrosine hydroxylase gene of S. mansoni (SmTH). The DNA fragments encoding SjTH were amplified by RT PCR. The PCR products were ligated to pGEM T easy vector and the inserts were confirmed by both restriction endonuclease digestion and DNA sequencing. Results The 480 bp DNA fragment was amplified by RT PCR from adult S. japonicum and was cloned into the pGEM T vector successfully .The DNA fragments was sequenced and analyzed. It had 87% homology with SmTH compared with completed cDNA of SmTH. Conclusion The incompleted sequence of SjTH is successfully amplified, which has 87% homology with SmTH. It will be used as a template for cloning and identification of the completed encoding sequence of SjTH by Rapid Amplification of cDNA Ends(RACE) PCR or other means for further research of this key molecule.