弓形虫核苷三磷酸水解酶-Ⅱ真核表达质粒的构建与鉴定  

Construction and identification of eukaryotic expression plasmid of Toxoplasma gondii NTPase-Ⅱ gene

作　　者：陈镭[1] 李成朋 陈弟[1] 赵徐寒晖 蒋凯[1] 林佳鑫 刘阳阳[2] 胡昕[2] 谭峰[3] 

机构地区：[1]温州医科大学第一临床医学院,温州325035 [2]温州医科大学生命科学学院检验医学院,温州325035 [3]温州医科大学基础医学院,温州325035

出　　处：《中国人兽共患病学报》2015年第6期519-521,526,共4页Chinese Journal of Zoonoses

基　　金：浙江省科技厅公益性项目(No.2014C33161);温州医科大学学生科研项目(No.wyx201301011)联合资助~~

摘　　要：目的构建弓形虫核苷三磷酸水解酶-Ⅱ(NTPase-Ⅱ)基因真核表达质粒pcDNA3.1(+)-NTPase-Ⅱ并在COS-7细胞中进行瞬时表达。方法以pBAD-HisB-NTPase-Ⅱ质粒为模板,PCR扩增NTPase-Ⅱ目的基因,将其克隆到pcDNA3.1(+)真核表达载体中,双酶切及测序鉴定重组质粒。阳离子脂质体法转染COS-7细胞并经SDS-PAGE和Western Blot检测目的蛋白的表达。结果经鉴定,弓形虫pcDNA3.1(+)-NTPase-Ⅱ核酸疫苗质粒构建成功。以脂质体法转染COS-7细胞后,转染细胞可成功地表达弓形虫NTPase-Ⅱ蛋白。结论证实了弓形虫NTPase-Ⅱ蛋白能在真核细胞中表达,为该基因的核酸疫苗研究提供了实验依据。We constructed the eukaryotic expression plasmid pcDNA3.1（＋）-NTPase-Ⅱ of Toxoplasma gondii and assess the expression of recombinant protein in COS-7cells.The prokaryotic expression plasmid pBAD-HisB-NTPase-Ⅱ constructed previously in our laboratory was used as templates to amplify the target gene by PCR.The resulting PCR products were cloned into the vector pcDNA3.1（＋）for construction of eukaryotic expression plasmid pcDNA3.1（＋）-NTPase-Ⅱ.Then,the plasmids were transfected into COS-7cells after identification with double enzyme digestion and sequencing.Finally,Western blot was performed to detect the expression of NTPase-Ⅱ in COS-7.The result revealed that Toxoplasma NTPase-Ⅱ could be expressed in COS-7cells when compared with empty pcDNA3.1（＋）control group.Taken together,the plasmid pcDNA3.1（＋）-NTPase-Ⅱ constructed successfully will play an important role in development of a potential gene vaccine.

关 键 词：刚地弓形虫 核苷三磷酸水解酶 真核表达 

分 类 号：R382.5[医药卫生—医学寄生虫学]