| 摘要: |
| 目的:考察人源神经生长因子信号肽序列是否具有介导β-内啡肽分泌性表达的作用,以及人源与鼠源神经生长因子信号肽的作用效率是否存在差异。方法:构建2个分别利用人或鼠的信号序列介导β-内啡肽分泌表达的真核表达载体pcDNA3.1-hEP和 pcDNA3.1-mEP;脂质体法转染NIH3T3细胞,转染48 h后分别收集细胞培养上清液、细胞,以及细胞呈单层生长的盖玻片。收集的上清液用RIA法测定β-内啡肽的浓度;收集的细胞提取总RNA,一步法RT-PCR检测融合基因的mRNA的转录;长有细胞的盖玻片处理后进行细胞免疫荧光染色。结果:RT-PCR的结果显示,所构建的融合基因在NIH3T3细胞内发生了表达,β-内啡肽主要分布在NIH3T3细胞的胞质内。pcDNA3.1-hEP和 pcDNA3.1-mEP分别转染NIH3T3细胞48 h后,细胞培养上清β-内啡肽的浓度分别是（280.33±24.16） pg/ml和（191.04±7.96） pg/ml,有显著的统计学差异（P<0.05）,且与空白对照之间均有显著的统计学差异（P<0.01）。结论:人源神经生长因子信号肽序列能够介导β-内啡肽蛋白的分泌性表达,且作用效率要优于鼠源信号肽。 |
| 关键词: 信号肽 神经生长因子 β-内啡肽 分泌性表达 |
| DOI：10.3724/SP.J.1008.2009.0378 |
| 投稿时间：2008-06-12修订日期：2009-03-06 |
| 基金项目:国家自然科学基金(30700788). |
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| Signal peptide of human nerve growth factor is better than signal peptide of mouse nerve growth factor in mediating secretory expression of beta-endorphin |
| XU Xue-wu, PEI Shu-jun, MIAO Xue-rong, YU Wei-feng* |
| (Department of Anesthesia, Eastern Hepatobiliary Hospital, Second Military Medical University, Shanghai 200438, China) |
| Abstract: |
| Objective:To clarify whether the signal peptide of human nerve growth factor can mediate secretory expression of beta-endorphin and whether there is difference between the efficiency of signal peptides from human and mouse nerve growth factor. Methods: Two kinds of eukaryotic vectors containing human or mouse signal sequence-mediated secretory expression of beta-endorphin were constructed.The culture supernatant and cells were collected 48 h after NIH3T3 cells were transfected by the two kinds of vectors, and the cover slips with single-layer cells was prepared. The concentration of beta-endorphin in the culture was determined by radio-immunoassay. The total RNA was extracted from cells and mRNA from fusion genes was assayed by RT-PCR. Cells on cover slips were subjected to immunofluorescence staining. Results: RT-PCR showed that the fusion genes were expressed in NIH3T3 cells; the expression of beta-endorphin was mainly in the cytoplasm of NIH3T3 cells. The concentrations of beta-endorphin in the supernatants 48 h after transfection with pcDNA3.1-hEP and pcDNA3.1-mEP were (280.33±24.16) pg/ml and (191.04±7.96) pg/ml (P<0.05), respectively, and they were significantly different from that of the blank control group (P<0.01). Conclusion: The signal sequence of human nerve growth factor can mediate the secretory expression of protein and the efficacy of human signal peptide is higher than that of mouse signal peptide. |
| Key words: signal peptide nerve growth factor beta-endorphin secretory expression |
