摘要
目的:构建针对E2F3基因的腺相关病毒干扰穿梭质粒载体,为进一步包装能够表达干扰序列的病毒奠定基础。方法:通过已经合成的pRNAT-U6.1-siE2F3/Neo干扰质粒载体,进行限制性内切酶Mlu酶切,将干扰片段克隆入线性质粒pAAV-MCS中,构建具有表达沉默E2F3基因的RNA干扰穿梭质粒。通过Bgl Ⅱ及Mlu单酶切,BamH1和HandⅢ双酶切、PCR鉴定及基因片段序列分析验证构建是否成功。结果:pAAV-siE2F3线性化后,片段大小约6 300bp;以Mlu为两边酶切位点设计的引物对重组质粒进行PCR,可获得的产物大小1 700bp;应用ITR专用引物进行重组质粒的PCR后证实本研究所构建的重组质粒ITR片段存在,大小约3 500bp,上述指标均与预期设计结果相符。基因测序结果表明插入序列无基因突变,序列完整。结论:穿梭质粒载体的成功构建,为进一步包装能够表达针对E2F3基因干扰序列的腺相关病毒,进一步研究E2F3基因在肿瘤中的功能奠定基础。
Objective： To construct shuttle plasmid vector of adeno-associated virus expressing small interferenceRNA. Methods： Lined pRNAT-U6.1-siE2F3/Neo plasmid was digested by restrictive endonucleaseMlu and the interfering component was cloned by polymerase chain reaction and inserted into linedpAAV-MCS plasmid to construct RNAi shuttle plasmid of AAV. The reconstructed RNAi plasmid was identifiedby electrophoresis after digestion with BgⅢ, Mlu, BamHI and HindⅢ. Polymerase chain reaction and gene sequenceanalysis can be used to confirm the accuracy of interference sequence. Results： Lined recombinantpAAV-U6.1-siE2F3-MCS vector fragment was of 6,300 base-pair. The fragment of recombinant plasmid vectorof adeno-associated virus identified by polymerase chain reaction was of 1,700 base-pair. Recombinant plasmidcontains inverted terminal repeat sequence and the fragment was of 3,500 base-pair. Sequencing analysisdemonstrated that the insertion sequence was exactly correct with no gene mutation. Conclusion： Successfulconstruction of AAV RNAi plasmid targeting E2F3 gene will facilitate the packaging of virus and the studyof E2F3 activities
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2009年第19期1117-1119,共3页
Chinese Journal of Clinical Oncology
基金
国家自然科学基金(编号:30700834)
天津市科技计划项目(编号:07ZCGYSF05200)
天津市高等学校科技发展基金计划项目(编号:20060122)
天津市卫生局科技基金资助(编号:05KYZ60)~~
