弓形虫ROP2基因的克隆与鉴定  

Cloning and Identification of ROP2 Gene of Toxoplasma gondii

作　　者：魏庆宽[1] 张佃波[1] 李瑾[1] 李桂萍[1] 王洪法[1] 崔勇[1] 柏雪莲[1] 付斌[1] 刘玉冰[1] 宰德富[1] 黄炳成 刘克义[1] 韩广东[1] 

机构地区：[1]山东省寄生虫病防治研究所,世界卫生组织淋巴丝虫病,绦/囊尾蚴病合作中心,济宁272033

出　　处：《中国寄生虫学与寄生虫病杂志》2006年第3期208-211,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：山东省自然科学基金(No.022130133)~~

摘　　要：目的体外扩增弓形虫棒状体分泌抗原2(ROP2)靶基因,构建真核表达载体pc-DNA3-ROP2。方法收集、纯化RH株弓形虫速殖子,提取基因组DNA;根据基因库ROP2基因序列设计合成1对引物,应用PCR扩增ROP2基因片段,回收纯化后克隆入TA载体质粒pUCm-T;用限制性内切酶EcoRⅠ、HindⅢ双酶切该重组子,将切下的ROP2基因在T4DNA连接酶作用下插入真核细胞表达载体质粒pc-DNA3,并进一步作双酶切、PCR及测序鉴定。结果以弓形虫基因组DNA为模板,PCR扩增出1.7kbROP2基因片段,克隆于pUCm-T载体中,再将ROP2基因亚克隆于真核表达载体质粒pc-DNA3,经筛选鉴定,构建pc-DNA3-ROP2重组质粒;测序结果显示,重组质粒包含了ROP2蛋白基因读码框内的完整序列,能完整表达ROP2的抗原蛋白。结论弓形虫ROP2基因片段,经TA克隆及亚克隆,构建弓形虫pc-DNA3-ROP2重组质粒。Objective To amplify ROP2 from the genomic DNA of Toxoplasma gondii RH strain and construct eukaryotic expression plasmid pc-DNA3-ROP2. Methods Tachyzoites of T. gondii RH strain were collected and depurated to obtain genome. A pair of primers was designed and synthesized according ROP2 gene sequence. The gene fragment encoding ROP2 was amplified from the genomic DNA of T. gondii RH strain by means of PCR. It was then reclaimed and purified, and inserted into cloning vector pUCm-T. The recon was cut by EcoR Ⅰ , Hind Ⅲ, and the inserted ROP2 gene fragment was subcloned into pc-DNA3 eukaryotic expression vector using T4DNA ligase, followed by further PCR identification, double digestion via restrictive enzymes, and sequencing. Results The amplified specific gene fragment of ROP2 was about 1.7 kb in length. The gene fragment cloned and subcloned into pc-DNA3 was correct, and the eukaryotic expression plasmid contained ROP2 gene fragment was successfully constructed. Conclusion The recombinant plasmid pc- DNA3-ROP2 was successfully constructed.

关 键 词：弓形虫 弓形虫棒状体抗原2 克隆 基因重组 

分 类 号：R382.5[医药卫生—医学寄生虫学]