【摘要】 目的寻找约氏疟原虫红外期发育相关基因,进行序列分析并推测其功能。方法分别提取约氏疟原虫感染大鼠(IL)和正常大鼠(UL)肝脏总RNA,根据疟原虫基因A+T含量高( >70%)的特点,设计可选择性扩增约氏疟原虫基因的G+C含量为40%的引物,采用差异显示PCR(differential display RT-PCR,DD-PCR)技术进行扩增,筛选差异片段,经TA克隆后测序并进行序列分析。结果找到6个与约氏疟原虫红外期发育相关基因(PyHs4、PyHs5、PyHs6、PyHs7、PyHs8、PyHs9);其中PyHs4与约氏疟原虫乙酰葡萄糖胺磷酸变位酶(phosphoacetylglucosamine mutase,AGM)具有相似性,PyHs5与约氏疟原虫红细胞膜蛋白3(erythrocyte membrance protein,PyHs5)具有同源性,PyHs6、PyHs7、PyHs8、PyHs9为功能未知基因。结论应用DD-PCR技术成功扩增了与约氏疟原虫红外期发育相关基因,为进一步的重组表达进功能和疫苗研究奠定基础。更多还原

【Abstract】 Objective To find the development-related genes of Plasmodium yoelii by sequence analysis and predict their functions during exoerythrocytic stage. Methods Total RNA was extracted from the liver of normal rat and Plasmodium yoelii infected rat followed by DD-PCR with the primer containing 40% G+C. The primer designed and synthesized accommodated the high adenine and thymidine rich genome of Plasmodium yoelii. The special bands of the PCR products were purified and cloned into plasmid T-vector. The recombinant plasmids were sequenced and analyzed. Results Six new genes (PyHs4, PyHs5, PyHs6, PyHs7, PyHs8, PyHs9) were related to the development of Plasmodium yoelii during exo-erythrocytic stage. In these genes, PyHs4 and PyHs5 had homology with phosphoacetyl glucosamine mutase (AGM) and erythrocyte membrance protein (EMP3) respectively. PyHs6, PyHs7, PyHs8, PyHs9 were all hypothetical genes with unknown function. Conclusion The developmental genes of Plasmodium yoelii have been amplified successfully by DD-PCR technique during exo-erythrocytic stage, which lays the foundation for the recombinant expression and new vaccine design.更多还原