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目的:构建人睾丸基因TDRG1的shRNA表达质粒,并研究其在NTERA-2细胞中的表达。方法:设计合成有短发夹结构靶位TDRG1基因的寡核苷酸,经退火成双链,克隆至载体pGPU6/GFP/Neo中,构建TDRG1shRNA表达载体,得到重组质粒TDRG1-shRNA486,TDRG1-shRNA738,TDRG1-shRNA921和一个阴性对照,使用Lipofectamine 2000转染shRNA至NTERA-2细胞,应用RT-PCR法检测转染后NTERA-2细胞TDRG1 mRNA的表达水平。结果:经测序证实,插入的DNA片段的序列与设计序列完全一致。同时筛选到转染TDRG1-shRNA486的NTERA-2细胞TDRG1基因的mRNA表达水平明显抑制。结论:成功构建了人类睾丸基因TDRG1的shRNA表达载体,TDRG1-shRNA在NTERA-2细胞内成功表达。
Objective: To construct shRNA expression plasmid of human testis gene TDRG1 and study its expression in NTERA-2 cells. Methods: The oligonucleotides of TDRG1 gene with short hairpin structure were synthesized and annealed to double strands. The TDRG1shRNA expression vector was constructed and the recombinant plasmids TDRG1-shRNA486 and TDRG1-shRNA738 , TDRG1-shRNA921 and a negative control. ShRNA was transfected into NTERA-2 cells using Lipofectamine 2000. The expression of TDRG1 mRNA in NTERA-2 cells was detected by RT-PCR. Results: Sequencing confirmed that the sequence of the inserted DNA fragment was exactly the same as the designed sequence. Meanwhile, the mRNA expression level of TDRG1 in NTERA-2 cells transfected with TDRG1-shRNA486 was significantly inhibited. Conclusion: The human testis gene TDRG1 shRNA expression vector was successfully constructed and TDRG1-shRNA was successfully expressed in NTERA-2 cells.