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目的构建以大鼠结缔组织生长因子(CTGF)和金属蛋白酶组织抑制因子1(TIMP-1)基因为靶点的短发夹RNA(shRNA)表达质粒。方法根据前期筛选出的对CTGF和TIMP-1基因最有效的RNA干扰靶位,各设计一对有短发夹结构的RNA干扰(RNAi)靶点序列,退火形成双链DNA,克隆到质粒载体psiRNA-h7SKGFPzeo;构建重组质粒载体psiRNA-GFP-CTGF和psiRNA-GFP-TIMP-1,并进行酶切与测序鉴定。结果酶切与序列测定均提示重组质粒psiRNA-GFP-CTGF和psiRNA-GFP-TIMP-1构建成功。结论成功构建靶向大鼠CTGF和TIMP-1最有效的RNAi靶位的shRNA表达质粒,为进一步探索肝纤维化基因治疗的新途径打下了实验基础。
Objective To construct a short hairpin RNA (shRNA) expression plasmid targeting rat connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase 1 (TIMP-1). Methods According to the targets of RNA interference targeting the most effective CTGF and TIMP-1 genes screened out in the early stage, a pair of RNA interference (RNAi) target sequences with short hairpin structure were designed and annealed to form double-stranded DNA, and cloned into the plasmid vector psiRNA-h7SKGFPzeo. The recombinant plasmid vector psiRNA-GFP-CTGF and psiRNA-GFP-TIMP-1 were constructed and verified by restriction enzyme and sequencing. Results Both digestion and sequencing showed that the recombinant plasmids psiRNA-GFP-CTGF and psiRNA-GFP-TIMP-1 were successfully constructed. Conclusion The successful construction of shRNA expression plasmids targeting rat CTGF and TIMP-1 RNAi targets lay the experimental foundation for further exploration of new ways of gene therapy for hepatic fibrosis.