论文部分内容阅读
目的:构建针对RhoA基因的shRNA表达载体并进行鉴定。方法:针对人RhoA的mRNA序列,设计并合成编码的shRNA的两条寡核苷酸序列,经退火成互补双链,再克隆至pGPU6/GFP/Neo质粒,构建重组体pGPU6/GFP/Neo-RhoA,进行酶切及测序鉴定,然后脂质体转染LoVo细胞。荧光显微镜观察绿色荧光蛋白的表达。结果:将合成的DNA序列退火后克隆到载体,酶切及测序证实质粒为所需的序列。结论:成功地构建了针对RhoA基因的shRNA表达载体,为下一步进行RNAi的相关研究奠定了基础。
Objective: To construct shRNA expression vector targeting RhoA gene and identify it. Methods: According to the mRNA sequence of human RhoA, two oligonucleotides encoding the shRNA were designed and synthesized. After annealing, the two strands were annealed and double cloned into pGPU6 / GFP / Neo plasmid to construct recombinant pGPU6 / GFP / Neo- RhoA, digested and sequenced, then transfected into LoVo cells. Fluorescence microscopy was used to observe the expression of green fluorescent protein. Results: The synthesized DNA sequence was annealed and cloned into the vector. Digestion and sequencing confirmed that the plasmid was the desired sequence. Conclusion: The shRNA expression vector targeting RhoA gene was successfully constructed, which laid the foundation for the further research on RNAi.