弓形虫可遗传及可诱导的RNAi载体系统的构建  

The Construction of the Inherited and Inducible RNAi Vector System for Toxoplasma gondii

作　　者：吴焜[1] 程璐[1] 王琼[1] 郝丽[1] 陈晓光[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院病原生物学系,广州510515

出　　处：《热带医学杂志》2008年第5期403-407,415,共6页Journal of Tropical Medicine

基　　金：国家自然科学基金(No.30671837);高等学校博士学科点专项科研基金(No.20069981003);广东省医学科研基金(No.B2005078)

摘　　要：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 for Toxoplasrna gondii to analyze the function of the genes. Methods Vector, pBSK-SAG1/5UTR-eGFP-SAG1/3UTR （pBSK-SAG1/GFP）, containing the GFP gene driven by the SAG1 promoter of T. gondii was constructed. The inducible RNAi vector, pBSK- HSPTO/SUTR-IntronC-HSPTO/3UTR, containing the inducible promoter, HSP70, was constructed. The fragment of SAG1/SUTR-eGFP-SAG1/3UTR in pBSK-SAG1/GFP vector was cloned into the vector of pBSK-HSP70/SUTR-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 to yield the vector pHANA-hairpin. The sequences and reversing sequences of SAG1 gene or BAG1 gene of T.gondii were amplified by PCR, and cloned into the vector pHANA-hairpin. The RNAi vectors targeted SAG1 gene, pHANA-hairpin/SAG1, or targeted BAG1 gene, pHANA-hairpin/BAG1, were constructed. Results The recombinant vectors were proved by endonuclease digestion and DNA sequencing. Conclusion The inherited and inducible RNAi vector system for T. gondii was constructed successfully.

关 键 词：弓形虫 RNA干扰 反向遗传学 载体 速殖子 

分 类 号：R382.5[医药卫生—医学寄生虫学]