表达E2F1小干扰RNA的逆转录病毒载体的构建

Construction of recombinant retroviral vector expressing E2F1 short interference RNA

目的 :建立一个表达E2F1小干扰RNA的靶向抑制E2F1表达的重组逆转录病毒载体。方法 :通过重组DNA技术 ,将设计好的两条互补寡核苷酸片段退火后与载体连接 ,利用载体上的BglⅡ和EcoRⅠ位点进行双酶切初步鉴定 ,重组载体应产生 3 3 2bp和 6182bp两个片段 ,选择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 :表达siRNA的重组逆转录病毒载体构建成功。结论 :重组逆转录病毒载体的成功构建为下一步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 targetedly suppressed E2F1 expression. Methods Using recombinant DNA techniques, 2 complementary 69bp oligonucleotides designed with hairpin loop for E2F1siRNA were annealed, and then inserted into the retrovirus expression vector pSIREN. Positive vectors were analyzed through digestion with BgL Ⅱ and EcoRⅠ, and obtained 2 fragments of 332bp and 6182bp by electrophoresion, and then DNA sequencing demonstrates the correct orientation and sequence. Results recombinant retroviral vector expressing short interference RNA (siRNA) had been constructed successfully.Conclusion Recominant retronral vertor may be used for further investigation of genetherapy.