DHFR基因RNA干扰质粒载体的构建及鉴定

摘要

Objective: To construct dyhidrofolate reductase gene( DHFR ) small interfering RNA( siRNA )and its expression vector,and explore the significance of DHFR inhibition in the treatment of platinum multidrug-resistance in ovarian cancer. Methods: We designed DHFR gene-targeted hairpin siRNA and then screened the best siRNA silence fragment, and inserted it into pGPU6/ GFP/Neo plasmid, which was identified by sequencing. Human malignant ovarian cancer SKOV3 cells were transfected with the constructed vector using lipofectin transfection method. Fluorescence photographs were taken. Results: Three groups of DHFR gene-targeted hairpin siRNA were designed, and were inserted into pGPU6/GFP/Neo vector after annealing. Vectors containing siRNA were right by sequencing. Fluorescence photographs showed that SKOV3 cells were transfected successfully by lipofectin transfection method, and PCR method was used to identify its interference effect. Then G418 method was adopted to screen the stable cell line. Conclusion: DHFR gene-targeted siRNA and its vector were successfully constructed. And one sequence with the highest inhibition efficiency was screened out. DHFR gene expression declined obviously after the constructed plasmid was transfected into SKOV3 cell.%目的:构建二氢叶酸还原酶(dyhidrofolate reductase,DHFR)基因RNA干扰质粒载体,为探讨抑制DHFR基因表达在卵巢癌铂类耐药治疗中的意义奠定基础.方法:设计DHFR基因靶向的发夹状siRNA,筛选出最佳siRNA沉默片段,退火后连接入pGPU6/GFP/Neo载体,转化后进行序列鉴定,脂质体转染SKOV3细胞株,并进行荧光摄像.结果:将退火后的双链寡核苷酸片段连接到pGPU6/GFP/Neo载体,测序结果正确.转染SKOV3细胞后,PCR鉴定干扰效果,G418筛选稳定细胞株.结论:成功构建针对DHFR基因的siRNA载体,找到了有效的干扰靶序列,转染SKOV3细胞后,DHFR基因的表达明显减弱.