人源性Jagged1 RNAi慢病毒载体的构建及鉴定

目的构建人源性Jagged1 RNAi慢病毒载体,并观察其对舌癌细胞中Jagged1的敲减作用。方法 GenBank检索人Jagged1信息,设计Jagged1靶点。BLAST比对同源性,合成双链DNAoligo。线性化的RNA干扰载体质粒pAJ-U6-shRNA-CMV-eGFP/PuroR,双酶切,与合成的双链DNAoligo连接,转化感受态细胞,选择阳性克隆进行鉴定。慢病毒三质粒系统共转染293T细胞,包装,纯化,并转染Tca8113以及Cal27细胞,测定转染效率。PCR及Western Blot检测Jagged1基因及蛋白表达变化。结果成功构建靶向Jagged1 RNAi载体质粒,并成功进行慢病毒包装。该系统可有效地感染Tca8113及Cal27细胞。细胞内Jagged1基因及蛋白表达均有明显下调。结论成功构建Jagged1 RNAi慢病毒载体,为下一步研究Jagged1在人舌鳞癌的中的作用机制奠定基础。

Construction and identification of the human Jagged1-targeted RNAi system on lentiviral vector

Objective To construct human Jagged1-RNAi lentiviral vector,and to observe the knocking-down effect in tongue cancer cells.Methods Human Jagged1 information was retrieved in GenBank and for designation of Jagged1 silence targets.After BLAST homology comparison test,synthetic double-stranded DNAoligo was constructed.Plasmid RNA interference pAJ-U6-shRNA-CMV-eGFP/PuroR was subsequently linearized,digested with restriction enzymes and recombined with the synthetic double-stranded DNAoligo.The recombinant plasmid was transfected into E.coli competent cells and selected for positive clones.293T cells was infected with the Jagged1 RNAi expression plasmid pAJ-U6-Jagged1-shRNA-CMV-eGFP/PuroR.Three lentiviral systems were co-transfected into 293T cells.Lentiviral vector packaged from 293T cells were purified.Tca8113 and Cal27 cells were transfected with the to assess the transfection efficacy.Real time PCR and Western Blot were used to detect intracellular Jagged1 gene and protein expression.Results The Jagged1-RNAi lentiviral vector was successfully constructed.Intensive green fluorescent cells can be seen in Tca8113 and Cal27 cells,suggesting effective infection.Intracellular Jagged1 gene and protein expression were significantly reduced according to Real time PCR and Western Blot in transfected cells.Conclusion The Jagged1-RNAi lentiviral vector is successfully constructed,providing foundation for further research of the mechanism of Jagged1 and Notch signaling pathway in the development of human tongue cancer squamous cell carcinoma.