弓形虫RNAi载体系统的构建
he Construction of the Inherited and Inducible RNAi Vector System of Toxoplasma gondii
目的 构建弓形虫可遗传及可诱导的RNAi载体系统，为弓形虫基因功能研究提供工具。方法 通过PCR和酶切连接，首先构建弓形虫主要表面抗原1(SAG1)基因启动子驱动的绿色荧光蛋白基因载体pBSK-SAG1/5UTR-eGFP-SAG1/3UTR(pBSK-SAG1/GFP)，然后构建以弓形虫热休克蛋白HSP70基因启动子驱动的反向重复序列RNAi载体pBSK-HSP70/5UTR-IntronC-HSP70/3UTR，将载体pBSK-SAG1/GFP中的SAG1/5UTR-eGFP-SAG1/3UTR片段克隆到载体pBSK-HSP70/5UTR-IntronC-HSP70/3UTR中形成载体pBSK-GFP-Hairpin，再将该载体中的GFP-Hairpin片段克隆到载体pHANA-0.5中形成弓形虫可遗传及可诱导的RNAi载体系统pHANA-hairpin。通过PCR分别扩增SAG1和缓殖子蛋白1(BAG1)基因的正向和反向序列，通过酶切连接，将正向和反向序列克隆到载体pHANA-hairpin中，分别构建靶向SAG1和BAG1基因的RNAi载体pHANA-hairpin/SAG1和pHANA-hairpin/BAG1。结果 酶切鉴定和测序结果表明成功构建载体pHANA-hairpin、pHANA-hairpin/SAG1和pHANA-hairpin/BAG1。结论 弓形虫可遗传及可诱导的RNAi载体系统成功构建，为下一步基因功能研究奠定基础。
Objective To construct the inherited and inducible RNAi vector system of Toxoplasma gondii to analyze the function of the genes. Method By the way of PCR, enzyme digestion and ligation, the vector, pBSK-SAG1/5UTR-eGFP-SAG1/3UTR (pBSK-SAG1/GFP), with the GFP gene drived by the SAG1 promotor of T. gondii, was constructed; and then the inducible RNAi vector, pBSK-HSP70/5UTR-IntronC-HSP70/3UTR, with the HSP70 gene promotor as a promotor, was constructed. The fragment of SAG1/5UTR-eGFP-SAG1/3UTR in pBSK-SAG1/GFP vector was cloned into the vector of pBSK-HSP70/5UTR-IntronC-HSP70/3UTR to construct pBSK-GFP-Hairpin vector,then the fragment of GFP-Hairpin in pBSK-GFP-Hairpin vector was cloned into pHANA-0.5 vector. Finally,the vector pHANA-hairpin was constructed. The sequences and reversing sequences of SAG1 gene or BAG1 gene of T. gondii were cloned by PCR, the PCR products were digested by the corresponding enzymes and ligatd into the vectors of pHANA-hairpin. The RNAi vectors targeted SAG1 gene, pHANA-hairpin/SAG1, or targeted BAG1 gene, pHANA-hairpin/BAG1,were constructed. Result The recombinant vectors were proved by endonuclease digestion and DNA sequencing. Conclusion The inherited and inducible RNAi vector system of T. gondii was constructed successfully.
王琼、程璐、陈晓光、郝丽、吴焜
分子生物学遗传学生物工程学
弓形虫RNA干扰反向遗传学载体速殖子
oxoplasma gondiiRNAireverse geneticsvectortachyzoite
王琼,程璐,陈晓光,郝丽,吴焜.弓形虫RNAi载体系统的构建[EB/OL].(2010-02-04)[2025-08-11].http://www.paper.edu.cn/releasepaper/content/201002-229.点此复制
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