靶向Galectin-1基因的siRNA真核表达载体构建及鉴定

Construction and identification of Galectin-1 targeting siRNA-expressing vector

目的:构建Galectin-1特异性siRNA真核表达载体psilencircle-G1si,转染细胞株U14,下调Galec-tin-1的表达。方法:运用生物信息学技术候选siRNA序列,合成SECs转染U14细胞,以荧光定量PCR快速筛选有效序列;siRNA序列插入siRNA真核表达质粒psilencircle,构建psilencircle-G1si并转染U14细胞,实时荧光定量PCR、Western blot检测Galectin-1表达量。结果:psilencircle-G1si酶切、测序表明与设计一致,实时定量PCR及western blot表明psilecircle-G1si能调低U14细胞Galectin-1的表达。结论:成功构建Ga-lectin-1特异性siRNA真核表达载体psilencircle-Glsi。

Objective：To construct Galectin-1 targeting siRNA expressing vector and down regulate Galectin-1 expression in U14 cell line.Methods： The series sequence of Galectin-1 targeting siRNA were found by using bioinformatics tools.The siRNA expression cassettes（SECs） was generated by PCR,then U14 cells were transfected by Galectin-1 targeting SECs.The efficient SEC was chosen by comparison of Galectin-1 mRNA level using real time fluorescent quantitative PCR（FQ-PCR） and establish the psilecircle-G1si according to the sequence of efficient SEC.U14 cell line which transfected psilecircle-G1Si was analyzed by FQ-PCR and Western blotting.Results： psilecircle-G1Si was identified by restriction enzyme analysis and DNA sequence analysis.FQ-PCR and Western blotting results have shown Galectin-1 expression in U14 cells was down-regulated.Conclusion： The Galectin-1 siRNA plasmid（psilencircle-G1Si） has been successfully constructed.It might provide a foundation for further study on the strategy to avoid tumor cells killing T lymphocytes.