摘要
目的构建针对PPARγ2基因siRNA重组逆转录病毒质粒载体,为进一步基因治疗奠定基础。方法选择RNA干涉靶序列,体外合成两段互补的寡核苷酸,通过与载体连接,转化大肠杆菌,扩增、纯化得到所需质粒,通过聚合酶链反应(PCR)琼脂糖凝胶电泳鉴定及利用载体上的BamHⅠ,C laⅠ和H indⅢ位点进行两次双酶切鉴定,重组载体应分别产生350bp,1100bp,5000bp和430bp,1100bp,5000bp六个片段,选择电泳及酶切正确的重组载体进行测序,保证siRNA的方向和序列正确。结果质粒中插入寡核苷酸的序列与设计的序列完全相符。结论成功构建了针对PPARγ2基因的siRNA重组逆转录病毒载体,为基因治疗的研究奠定了基础。
Objective To establish a recombinant retroviral vector expressing short interference RNA （siRNA） and study the function of PPARγ2 with RNA interference technology. Methods Using recombinant DNA techniques, 2 complementary 82bp oligonucleotides designed with hairpin loop for siRNA were annealed, and then inserted into the retrovirus expression vector pSINsi - hU6. Positive vectors were analyzed through digestion with BamH Ⅰ , Cla Ⅰ , Hind Ⅲ, and obtained 6 fragments of 350bp, 1100bp, 5000bp and430bp, 1100bp,5000bp by eleetrophoresion. The plasmid was identified by PCR, and then DNA sequencing demonstrated the correct orientationand sequence. Results Recombinant retroviral vector expressing short interference RNA （siRNA） had been constructed successfully. Conclusion Reeominant retronral vertor may be used for further investigation of gene therapy.
出处
《中原医刊》
2006年第20期17-19,共3页
Central Plains Medical Journal
