论文部分内容阅读
目的构建针对人MAGE3基因的小干扰RNA(siRNA)及其表达载体,转染细胞后观察其对MAGE3基因的干扰作用。方法设计MAGE3靶向的发夹状siRNA,依据设计合成两条互补的寡核苷酸链,退火后连接入pSUPER.neo载体,转化扩增后进行序列测定。用脂质体包裹转染胃癌细胞BGC823,采用RT-PCR检测MAGE3基因mRNA表达水平的变化。结果把针对MAGE3基因的siRNA的双链寡核苷酸片段克隆到pSUPER.neo载体,经过酶切鉴定与测序,结果正确;稳定转染人胃癌细胞BGC823后,RT-PCR检测显示,MAGE3基因的表达水平明显降低。结论成功构建了针对MAGE3基因的siRNA载体,转染细胞后可抑制MAGE3基因表达。
Objective To construct small interfering RNA (siRNA) targeting human MAGE3 gene and its expression vector, and to observe its interference effect on MAGE3 gene after transfection. Methods MAGE3 targeted hairpin siRNAs were designed and synthesized according to the design of two complementary oligonucleotide strands. After annealing, they were ligated into pSUPER.neo vector, and transformed and amplified for sequencing. The gastric cancer cell BGC823 was transfected with liposome, and the mRNA expression of MAGE3 was detected by RT-PCR. Results The double-stranded oligonucleotide fragment of siRNA against MAGE3 gene was cloned into pSUPER.neo vector and verified by restriction enzyme digestion and sequencing. The result was correct. After transfection of human gastric cancer cell line BGC823, RT-PCR results showed that MAGE3 gene The expression level was significantly reduced. Conclusion siRNA vector targeting MAGE3 gene was successfully constructed and transfected into cells could inhibit MAGE3 gene expression.