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目的:构建针对CGRP基因的shRNA载体并进行鉴定。方法:针对小鼠CGRP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,再克隆至质粒,构建重组体pLLU2G/CGRP,转化至stbl3细菌,挑选阳性克隆测序鉴定。慢病毒包装后,脂质体转导至neuro2A,显微镜观察荧光蛋白的表达。结果:测序证实质粒为所需的序列,脂质体转导至neuro2A 48 h后可见绿色荧光蛋白,转导率达90%。结论:成功地构建了针对CGRP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。
AIM: To construct shRNA vectors targeting CGRP gene and identify it. Methods: According to the mRNA sequence of mouse CGRP, two oligonucleotides encoding shRNAs were designed and synthesized. After annealed into complementary double-stranded DNA, the recombinant plasmid pLLU2G / CGRP was constructed and transformed into stbl3 bacteria. Positive clones were identified by sequencing. After lentivirus packaging, the liposomes were transduced to neuro2A and the expression of the fluorescent protein was observed microscopically. Results: Sequencing confirmed that the plasmid was the desired sequence. The green fluorescent protein was seen after liposome transfection to neuro2A for 48 h, and the transduction rate was 90%. Conclusion: The shRNA expression vector targeting CGRP gene was successfully constructed, which laid the foundation for the further research on RNAi.