Construction of Transforming Growth Factor-β_1siRNA Expression Vector

Objective To construct a plasmid vector of RNA interference(RNAi) of transforming growth factor β1gene(TGF-β1),for the radiation-induced lung injury and other diseases,high expression of TGF-β1to provide a new method of gene therapy.Methods According to human TGF-β1mRNA sequence as a template,design and synthesis of oligonucleotides corresponding,then DNAsegment was gained through annealing after chemosynthesis,and then was cloned to pRNAT vector.The recombinant TGF-β1 siRNA expression vector was evaluated by using enzyme cutting.At last,the constructed plasmid TGF-β1-specific siRNA vector was transfected into human embryonic lung fibroblast HFL-I cells,and its effect on TGF-β1mRNA expression was observed by fluorescence quantitative PCR and ELISA,apoptosis by flow cytometry.Results Digestion and sequencing showed that the target has been accurately oligonucleotides cloned into plasmid vector linear pRNAT,compared with the control,TGF-β1mRNA level and protein level was significantly inhibited the expression levels of statistical significant difference(P0.05).Human embryonic lung fibroblast cells significantly increased(P0.05).Conclusion Success has constructed the person TGF-β1gene siRNA material particle carrier,has laid the foundation regarding the radioactive lung damage as well as other TGFβ1high expression disease's gene therapy.