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目的:构建RKIP真核表达质粒,并检测其在PC12细胞中的表达。方法:从大鼠背根神经节细胞中提取总RNA,应用巢式PCR技术,扩增获得RK IP基因编码序列片段,克隆入真核表达载体pcDNA3.0,对重组质粒进行酶切和测序鉴定后,以Vigofect非脂质体介导法转染至PC12细胞,通过Western blot检测RKIP蛋白的表达。结果:酶切和测序结果证明pcDNA3.0-RKIP重组质粒的DNA序列完全正确,将其转染PC12细胞后,RKIP蛋白表达明显增加。结论:pcDNA3.0-RKIP真核表达质粒构建成功,并能在PC12细胞内表达,为深入研究RKIP的神经生物学功能提供了实验基础。
Objective: To construct RKIP eukaryotic expression plasmid and test its expression in PC12 cells. Methods: Total RNA was extracted from rat dorsal root ganglion cells. The amplified fragment was cloned into eukaryotic expression vector pcDNA3.0 by using nested PCR. The recombinant plasmid was digested and sequenced After transfected into PC12 cells by Vigofect non-liposome-mediated method, the expression of RKIP protein was detected by Western blot. Results: The DNA sequence of pcDNA3.0-RKIP recombinant plasmid was proved to be completely correct by restriction enzyme digestion and sequencing. The expression of RKIP protein in PC12 cells was significantly increased after transfection. Conclusion: The pcDNA3.0-RKIP eukaryotic expression plasmid was successfully constructed and expressed in PC12 cells, which provided the experimental basis for further study on the neurobiology function of RKIP.