适用于shRNA表达研究的克隆载体构建

Construction of a cloning vector appropriate for shRNA expression study

目的:建立一种适用于shRNA表达研究的克隆载体。方法:通过PCR扩增一段含有自杀基因ccdB和卡那霉素抗性基因的DNA序列,将其双酶切后克隆至shRNA表达载体pSilencer3.1-H1neo中,得到重组体pSilencer3.1-ccdB。运用此重组体构建荧光素酶的shRNA表达载体并设立对照进行非重组背景的结果比较。结果:成功建立了pSilencer3.1-ccdB克隆载体。应用此载体构建荧光素酶的shRNA的表达载体,结果显示完全消除了非重组背景。结论:建立了适用于shRNA表达研究的克隆载体,为进一步研究siRNA的基因功能奠定了基础。

Objective： To establish a novel vector with low non-recombinant background and high success rate of cloning and thus suitable for shRNA expression study. Methods： The DNA sequence containing ccdB suicide gene and kanamycin resistance gene was amplified by using PCR and cloned into the shRNA expression plasmid pSilencer3.1-H1 neo that was previously treated with appropriate restriction endonucleases. The power of the resulting vector pSilencer3.1-ccdB was evaluated by cloning the luciferase shRNA expression sequence. As control, the original parent vector pSilencer3.1-H1 was also employed to clone the sequence. Results：The pSilencer3.1-ccdB vector dramatically increased the success rate of cloning by removing the non-recombinant background as compared with the control vector. Conclusion： This study provides a useful tool that will prove its efficacy in the studies of gene function using RNA interference techniques.