弓形虫ROP18基因重组蜥蜴利什曼原虫的构建及鉴定  

Construction and Identification of Toxoplasma gondii ROP18 in Transgenic Leishmania tarentolae

作　　者：李博[1] 魏峰[1] 刘全[2] 马洪霞 许越[1] 金洪涛[2] 商立民[2] 

机构地区：[1]吉林农业大学生命科学学院,吉林长春130118 [2]军事医学科学院军事兽医研究所,吉林省人兽共患病预防与控制重点实验室,吉林长春130122 [3]公主岭市红十字血站,吉林四平136100

出　　处：《动物医学进展》2012年第2期1-4,共4页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31072127/31001057)

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。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.

关 键 词：蜥蜴利什曼原虫 弓形虫ROP18基因 重组蛋白 

分 类 号：S852.72[农业科学—基础兽医学]