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.
出处
《热带医学杂志》
CAS
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)
