两种不同肝纤维化相关细胞因子shRNA重组质粒的构建

摘要

Objective To construct short hairpin RNA (shRNA ) expression plasmid targeting at genes of rat connective tissue growth factor (CTGF) or tissue inhibitor of metalloproteinase 1 (TIMP1).Methods According to the most effective RNA interference sequences of rat CTGF gene and TIMP-1 gene screened out in the previous experiments, two pairs of CTGF and TIMPlgenespecific shRNA were designed and synthesized.After primer annealing, they were inserted into plasmid psiRNAh7SKGF-Pzeo and named as psiRNA-GFP-CTGF or psiRNA-GFP-CTGF, respectively.The two recombinant plasmids were confirmed by restriction enzyme digestion and sequencing.Results Restriction enzyme digestion and sequencing showed that the two doublestranded DNA fragments were inserted correctly into psiRNA-h7SKGFPzeo vectors as expected, respectively.Conclusion Two shRNA recombinant plasmids were constructed successfully, this would establish an experimental foundation for further exploring of new ways in gene therapy for hepatic fibrosis.%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.