摘要
目的构建抑制黑色素瘤抗原-1(MAGE-1)的siRNA表达载体,鉴定其在人恶性胶质瘤细胞系SHG-44细胞中对MAGE-1基因表达的干涉作用。方法化学合成2对编码短发夹RNA序列的靶向MAGE-1基因寡核苷酸链,克隆到经BglⅡ、HindⅢ双酶切的pSUPER载体上,重组构建RNA干涉(RNAi)质粒载体。利用逆转录多聚酶链反应(RT-PCR)、流式细胞术和荧光显微镜,检测经稳定转染后SHG-44细胞中MAGE-1的表达,以了解siRNA的干扰效果。结果重组构建的pSUPER-MAGE-1载体经双酶切电泳及插入基因片段序列分析,表明寡核苷酸链成功插入到预计位点,并且序列与预期完全一致。稳定转染后G418筛选出的SHG-44多克隆细胞MAGE-1的表达经RT-PCR、流式细胞术和荧光显微镜检测,2对siRNA均有较明显的干涉作用。结论成功构建了针对MAGE-1基因的siRNA表达载体,抑制SHG-44细胞中的MAGE-1分子的表达。
Objective To study the inhibition of expression of melanoma-associated antigen-1 （MAGE-1） by RNA interference （RNAi） in SHG-44 human glioma cell line. Methods Sixty-four base-pair oligonucleotides encoding for hairpin RNA which targeted MAGE-1 gene were chen-fically synthesized and annealed, pSUPER vector was linearized with BglⅡand Hind Ⅲ. Annealed oligonucleotides were inserted into the treated pSUPER to construct RNAi plasmid （pSUPER-MAGE-1）. Expression of MAGE-1 was detected in SHG-44 cell by reverse transcriptase- polymerase chain reaction （RT-PCR） after acquiring the stable transfectant cell line. Results Recombinant pSUPER-MAGE-1 vector was identified and confirmed by sequencing analysis. The results demonstrated that 64 bp had been inserted into the expected site and the insertion sequence was correct. The expression of MAGE-1 in SHG- 44 detected by RT-PCR, flow cytometry and fluorescence microscope was greatly downregulated by these two siRNA respectively. Conclusion pSUPER-MAGE-1 RNAi system can successfully inhibit the expression of MAGE-1 in SHG-44 and be able to facihtate the study on the function of MAGE-1.
出处
《中华神经外科疾病研究杂志》
CAS
2006年第2期106-110,共5页
Chinese Journal of Neurosurgical Disease Research
基金
国家自然科学基金资助项目(39970752)
