人源与鼠源神经生长因子信号肽引导β-内啡肽分泌表达的差异

Signal peptide of human nerve growth factor is better than signal peptide of mouse nerve growth factor in mediating secretory expression of beta-endorphin

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pcD-NA3.1-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mR-NA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。

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 eukaryotie vectors containing human or mouse signal sequence-mediated secretory expression of beta-endorphin were constructed. The culture supernatant arid 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.