犬新孢子虫NcAMA1基因重组腺病毒穿梭质粒的构建及其真核表达  

Construction and eukaryotic expression of the recombinant adenovirus shuttle plasmid with NcAMA1gene of Neospora caninum

作　　者：郭焕平[1] 贾立军[1] 于龙政[1] 薛书江[1] 高洋[1] 李男礼[1] 张守发[1] 

机构地区：[1]延边大学农学院动物医学系,吉林延吉133002

出　　处：《中国兽医科学》2014年第3期282-286,共5页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31160501;31360605);吉林省重点科技攻关项目(20140204078NY);吉林省青年科研基金项目(201201076)

摘　　要：为构建犬新孢子虫NcAMA1基因重组腺病毒穿梭质粒,并在真核细胞中表达,以重组质粒pVAX1-NcAMA1为模板,PCR扩增了NcAMA1基因,以此构建了pMD18T-NcAMA1重组克隆质粒;对该重组克隆质粒进行双酶切鉴定后,将其亚克隆至腺病毒穿梭载体pCR259中。应用脂质体介导转染法将经PCR鉴定和酶切鉴定正确的pCR259-NcAMA1重组穿梭质粒转染293细胞,应用IFAT和Western-blot技术检测了AMA1基因在293细胞中的表达情况。结果显示,扩增的NcAMA1基因长度为1 695bp,构建的重组腺病毒穿梭质粒pCR259-NcAMA1能在293细胞中获得瞬时表达,表达蛋白的分子质量约为68ku。To construct recombinant adenovirus shuttle plasmid with Neospora caninum NcAMA1 gene and to express the recombinant plasmid in eukaryotic cells, using the constructed eukaryotic recombi- nant plasmid pVAX1-NcAMA1 as a template, NcAMA1 gene was amplified by PCR, and the recombinant plasmid pMD18T-NcAMA1 was consturcted. The digested NcAMA1 gene was subcloned into adenovirus shuttle vector pCR259 digested with the same enzymes. The NcAMA1 recombinant adenovirus shuttle plasmid pCR259-NcAMA1 was then transfected into 293 cells by liposome. The expressed product in 293 cells was analyzed by IFAT and Western-blot. In result,the amplified gene NcAMA1 was 1 695 bp in size, the recombinant adenovirus shuttle plasmid pCR259-NcAMA1 was constructed successfully, and the recombinant plasmid transiently expressed in 293 cells,and the recombinant protein was about 68 ku in a mo- lecular mass.

关 键 词：犬新孢子虫 NcAMA1基因 腺病毒穿梭质粒 表达 

分 类 号：S852.723[农业科学—基础兽医学]