基质金属蛋白酶抑制剂-1基因的RNA干扰载体构建及其抑制效应

目的构建含有基质金属蛋白酶抑制剂-1(TIMP-1)基因特异性RNA干扰序列的慢病毒vshRNA载体,筛选有效靶点序列。方法通过在线软件设计TIMP-1基因的RNA干扰(RNAi)序列,按siRNA设计的优化原则筛选靶序列,构建含有TIMP-1基因特异性慢病毒vshRNA载体。将重组pEGFP-N1-3FLAG-TIMP-1质粒和慢病毒质粒pGCSIL-GFP-shRNA共转染293T细胞,转染48 h后收集细胞,抽提总蛋白进行Western Blot检测,筛选抑制效应较好的质粒。结果依次通过在线软件设计序列、优化筛选、BLAST比对等方法,选择4条作为靶序列,又经Western Blot检测筛选出抑制效应较好的1条序列,其在TIMP-1基因的173～192 nt处。结论成功构建了具有抑制效应的TIMP-1基因特异性RNA干扰序列的慢病毒vshRNA载体,筛选出抑制TIMP-1表达的序列,为抗肝纤维化研究奠定了基础。

Construction of the RNA interference vector of tissue inhibitor of matrix metalloproteinase-1 gene and its inhibitory effects

Objective To construct a lentivirus vshRNA vector harboring the specificity interference gene sequence of the tissue inhibitor of matrix metalloproteinase-1(TIMP-1) and screen the effective target sequence.Methods Firstly RNA interference sequence of TIMP-1 gene was designed using online software and then a lentivirus vshRNA vector including the specificity interference gene sequence of TIMP-1 was constructed after the target sequence was screened according to the principle of optimization based on small interfering RNA.The recombination pEGFP-N1-3FLAG-TIMP-1 plasmid was transfected together with the lentivirus pGCSIL-GFP-shRNA plasmid into 293 T cells and Western Blot was performed to detect the proteins extracted from these cells 48 hours later.Results Four target sequences were selected by the online software,screening and BLAST comparing,and one of them was found bearing the most effective inhibitory effect than the others by western blot.Its location was at 173-192nt in the TIMP-1 gene.Conclusion An inhibitory lentivirus vshRNA vector harboring the gene specificity interference sequence of TIMP-1 was successfully constructed and a sequence which has an inhibitory effect to the expression of TIMP-1 gene was screened,and laid the foundation for anti-fibrosis therapy.