人神经生长因子信号肽介导β-内啡肽的分泌表达

Signal Peptide from Human Nerve Growth Factor Mediated Secretory Expression of β-endorphin

目的：构建人神经生长因子信号肽与人β-内啡肽融合基因的真核表达载体，研究人神经生长因子信号肽介导β-内啡肽的分泌表达。方法：取得人基因组后，PCR法获取人的神经生长因子信号肽部分序列及人β-内啡肽序列；通过SOE—PCR法将两段DNA序列连接，然后插入到真核表达载体内，测序正确后扩增转染级的真核表达载体。表达载体脂质体法转染NIH3T3细胞，转染后48—72h收集细胞及培养上清，RT—PCR法检测融合基因的转录，RIA法测定细胞外β-内啡肽的浓度。结果：成功构建全人源的分泌型表达β-内啡肽的真核表达载体，DNA序列经测序完全符合实验设计；融合基因能够顺利地得到转录并进行表达翻译，在细胞培养上清中可检测到其产物。结论：构建的真核表达载体能够分泌表达人β-内啡肽，提示人神经生长因子信号肽序列能够发挥其介导蛋白产物分泌表达的作用。

Objective： To construct the eukaryotic vector expressing the fusion gene generated with the signal pepfide from human nerve growth factor and human β- endorphin, and to study its secretory expression mediated by the signal peptide from human nerve growth factor. Methods： After human geneme DNA obtained, the signal peptide sequence from human nerve growth factor and human β-endorphin were got respectively by PCR. The whole fragment, constructed by hgation of these above two fragments by SOE- PCR, was subcloned into the eukaryofic vector. The generated vector was upscaled to produce transfection- grade vector after it was sequenced right. NIH3T3 cells were transfected by produced vectors according to the manual of Lipofectamine2000. The transcfipted RNA of fusion gene was detected by RT- PCR and the endorphin concentration in cell culture media was measured by RIA between 48 to 72 hours after transfecfion. Results： The eukaryofic vector secretorily expressing human beta- endorphin was successfully constructed. The DNA sequence accorded with the designed sequence completely. The fusion gene was successfully transcripted and expressed. The expression product was detected in the cell culture media. Conclusion： The constructed eukaryofic vector can secretorily express human beta- endorphin. The selected signal peptide from human nerve growth factor can mediate the exeeytosis of expression product.