弓形虫棒状体蛋白ROP2基因的克隆与表达  

Clone and expression of rhpotry protein ROP2 from Toxoplasma gondii

作　　者：李文姝[1] 陆惠民[2] 闵太善[3] 黄伟达[3] 

机构地区：[1]温州医学院微生物免疫学教研室,温州325032 [2]苏州大学基础医学院 [3]复旦大学生命科学院生化与分子生物学实验室

出　　处：《中国人兽共患病杂志》2005年第10期883-886,共4页Chinese Journal of Zoonoses

摘　　要：目的从弓形虫RH株总DNA中克隆棒状体蛋白ROP2基因片段,克隆至表达载体pET22b中,导入大肠杆菌中表达。方法采用半巢式PCR扩增法从基因组DNA中扩增编码ROP2基因片段,克隆至质粒pUC119中,经PCR和酶切鉴定后,进行DNA序列测定,并以SacⅠ/HindⅢ双酶切克隆至表达载体pET22b上,重组质粒pET22b-ROP2转化大肠杆菌BL21-Codon Plus(DE3)-RIL菌株中,经IPTG诱导表达。结果从弓形虫RH株总DNA中扩增到1.0kb的ROP2基因片段,构建成功重组质粒pUC119-ROP2,经DNA序列分析与已报道序列基本一致,重组质粒pET22b-ROP2在大肠杆菌中表达,产生一分子量约为45kDa的预期重组蛋白。结论克隆到的弓形虫棒状体蛋白ROP2在大肠杆菌中得到表达,构建成功的重组质粒pUC119-ROP2可用于下一步多基因融合的克隆操作。To clone and express the gene coding for rhoptry protein ROP2 from the total DNA of Toxoplasma gondii of RH strain. The gene fragment encoding ROP2 was amplified by PCR from the total DNA of T. gondii of RH strain. And the gene of ROP2 was subcloned into the plasmid pUC119, After the identification by PCR and restriction enzyme cleavage, DNA sequence was determined. The Sac Ⅰ/Hind Ⅲdigested fragment of pUC119-ROP2 was inserted into the same site of expression vector pET22b. The recombinant plasmid of pET22b-ROP2 was transformed to a bacterium BL21-Codon Plus（ DE3）-RIL and was expressed under the induction of IPTG. The results showed that the gene fragment encoding 1.0kb ROP2 was obtained from total DNA of T. gondii of RH strain by PCR. The recombinant palsmid pUC119-ROP2 was constructed, and the aligment chart of the ROP2 DNA sequence between the obtained ROP2 and the reported ROP2 from gene bank showed that there were no significant difference from each other. The recombinant plasmid pET22b-ROP2 in E. coli expressed a molecular weight of 45kDa fusion protein. It is concluded that the recombinant plasmid pCU119-ROP2 which was constructed successfully could provid the necessity for the development of conjugated antigen of T. gondii.

关 键 词：弓形虫 棒状体蛋白ROP2 克隆表达 

分 类 号：R382.5[医药卫生—医学寄生虫学]