【摘要】 目的克隆用于人DNA聚合酶β(DNApolymerasebeta,Polβ)基因靶向RNA干扰的双链RNA(doublestrandRNA,dsRNA)寡核苷酸绿色荧光蛋白C1载体重组子“pEGFPC1pU6dsRNA”,为进一步研究人Polβ基因在环境化学污染物所致的DNA损伤修复中的作用机制作准备。方法依据Genbank中人polβ基因的cDNA序列,运用dsRNAoligonucleotidedesigner设计出用于RNA干扰用的dsRNA寡核苷酸序列,并以化学方法合成之。通过重组DNA技术将合成好的dsRNA克隆到含人pU6启动子的pSIRENRetroQ逆转录病毒载体上,以此重组子转化感受态E.coliDH5α,经氨卞青霉素筛选后进行扩增,并抽提质粒。运用EcoRⅠ和BglⅡ消化并回收“pU6dsRNA”目的片段,再将“pU6dsRNA”亚克隆到pEGFPC1上,获“pEGFPC1pU6dsRNA”重组子,并进行酶切鉴定和序列分析。结果经酶切鉴定发现,“pEGFPC1pU6dsRNA”载体重组子处于预期条带位置,序列分析显示与所设计的目的片段序列完全相符。结论作者克隆了用于人Polβ基因靶向RNA干扰的dsRNA绿色荧光蛋白C1载体重组子“pEGFPC1pU6dsRNA”,为进一步研究Polβ基因功能作好了准备。更多还原

【Abstract】 Objective To clone the “pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference, to provide research tool for the study on the function of DNA polymerase beta in repairing of human DNA damaged by environmental chemical pollutants(ECPs). Methods According to the gene sequence of polymerase beta cDNA published in Genbank, double strand RNA(dsRNA) sequence which was used in RNA interference was designed by dsRNA oligonucleotide designer and synthesized by chemical methods. DNA recombination technology was used to insert the up related dsRNA sequence into the vector of pSIREN-RetroQ, and then the “pSIREN-RetroQ-dsRNA" recombinant was obtained. After E.coli DH5α was transformed with the “pSIREN-RetroQ-dsRNA" recombinant and screened with ampicillin for positive clones, plasmid was extracted and digested by EcoRⅠand BglⅡ, the fragment of“pU6-dsRNA"was purified. And then the “pU6-dsRNA"fragment was cloned into the vector of pEGFP-C1 by recombination technology, the recombinant of “pEGFP-C1-pU6-dsRNA" was obtained and identified by restriction endonuclease analysis and sequencing. Results The “pEGFP-C1-pU6-dsRNA" recombinant lied in the predicted band, and the sequence of insert was identical to the designed target fragment. Conclusion The “pEGFP-C1-pU6-dsRNA" recombinant was successfully cloned for human DNA polymerase beta RNA interference, it was an important research tool for the further study.更多还原