靶向EGFR基因siRNA腺病毒载体构建及其对EL-4影响

Construction of the Adenovirus Expressing Mice EGFR SiRNA and its Effect on the Mice EL-4 Cell Line

目的构建含EGFR基因siRNA腺病毒载体,并研究其对EL-4细胞株中EGFR基因表达的影响。方法合成双链寡核苷酸并克隆入pShuttleH1载体,pShuttleH1-siEGFR线性化后电转化到含pAdEasy-1的BJ5183感受态细菌中获取重组质粒,脂质体转染293细胞获取重组腺病毒Ad-siEGFR;TCID50法测定病毒效价;利用得到的重组腺病毒感染鼠EL-4细胞株,采用Western-blot检测感染前后EGFR蛋白表达水平的变化,观察AdH1/siEGFR对EL-4细胞EGFR表达的影响。结果成功构建EGFRsiRNA腺病毒载体,该载体可明显抑制EL-4细胞中EGFR蛋白的表达。结论构建的AdH1/siEGFR腺病毒能有效抑制EGFR基因在EL-4细胞株中的表达,为胸腺瘤基因治疗提供了新的方法和材料。

Objective To construct the incompetent- replication adenovirus expressing mice EGFR siRNA and to investigate its effect on EL - 4 cells. Methods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was cloned into the pShuttleH1 vector. Linearized pShuttleH1 - siEGFR was transformed into E. coli BJS183 cells containing backbone plasmid pAdEasy - 1 by electroporation. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad - siEGFR. The ti- ters of adenovirus were determined using the specific 50% tissue culture infection dosage（TCID50） method. Result Ninety - six hours after Ad - siEGFR transfection into EL - 4 cells, the expression of EGFR protein was significantly decreased. Conclusion The recombinant adenovirus expressing mice EGFR siRNA were successfully constructed. It provides a new method for use in gene therapy for thymic tumor.