靶向Bcl-2基因转录shRNA重组质粒的构建和序列分析

Construction and Sequence Analysis of the Transcriptional Small Hairpin RNA Recombinant Plasmid Targeting Bcl-2 Gene

[目的]利用RNA干扰技术,以Bcl-2为靶基因,设计构建重组体,并进行序列分析,探索肿瘤基因治疗的新途径。[方法]设计有小发夹结构的两条DNA序列,经退火形成互补双链,克隆至转录载体Pgenesil-1上构建重组体,转化DH5a菌株,提取质粒行酶切鉴定后,进行测序分析。[结果]将合成的DNA片段退火后克隆至载体上,经酶切及序列鉴定为目的序列,靶向Bcl-2基因转录shRNA重组质粒构建成功。[结论]靶向Bcl-2基因转录shRNA重组质粒的成功构建,为下一步利用该重组体干扰肿瘤细胞中Bcl-2的mRNA转录,探索肿瘤基因治疗的新途径奠定基础。

[Purpose] To study the new approach of tumor gene therapy by constructing recombinant using Bcl-2 as targeting gene with the help of RNA interfering and by conducting sequence analysis. [Method] Two designed DNA sequences with small hairpin structure were cloned into the transcripted carrier Pgenesil-1 in order to construct recombinant, and the recombinant was transformed into DH5a strains.Then the sequence analysis was carried out after the recombinant plasmid was identified by enzyme restriction. [Results] The synthesized DNA fragment was cloned into the carrier after annealing. It was identified as an aim sequence by enzyme restriction and sequence analysis. The recombinant pasmid targeting Bel-2 gene was successfully constructed and the aim sequence was obtained. [Conclusion] Successfully constructing the recombinant plasmid provides the scientific base for searching a new gene therapy method for cancer.