【摘要】 用PCR方法从弓形虫RH株全基因组中扩增ROP18基因,将其克隆至pMD18-T载体,经测序分析后,构建真核表达穿梭质粒pLEXSY-neo2-ROP18;利用电转染技术将外源基因表达盒转入蜥蜴利什曼原虫,G418筛选阳性克隆;阳性克隆分离培养后提取基因组,用ROP18特异性引物及同源重组鉴定引物进行重组利什曼原虫PCR鉴定,结果阳性虫体中扩增出特异性目的条带;应用鼠抗弓形虫多抗血清以Western blot印记法从蛋白水平进行重组蛋白检测,证明目的蛋白在蜥蜴利什曼原虫中得到表达。更多还原

【Abstract】 In this study,the ROP18 gene of T.gondii was amplified by PCR method from T.gondii RH strain genome,and cloned into eukaryotic expression vector(pLEXSY-neo2) to construct the recombinant plasmid pLEXSY-neo2-ROP18.The plasmid pLEXSY-neo2-ROP18 was transfected by electroporation into the Leishmania tarentolae genome,and positive clones were screened by G418.The positive and negative control parasites’ genomes were extracted and used to amplify ROP18 fragment using specific primers.The results showed that specific target band could be detected in the positive parasites.Western blot analysis suggested the expressed protein can be recognized by anti-histidine antibody,and no target band in negative control was detected.So the target protein was expressed successfully in Leishmania tarentolae.更多还原