人表皮细胞生长因子基因真核表达质粒的构建

Construction of the eukaryotic expression plasmid for human epidermal growth factor gene

目的：进行外源性基因真核表达的系统构建。方法：以肾组织ｍＲＮＡ为模板进行逆转录ＰＣＲ扩增人表皮细胞生长因子（ｈＥＧＦ）信号肽基因；将扩增的特异性ＤＮＡ条带克隆于ｐＢＫ－ＣＭＶ质粒中进行序列测定；再将已扩增的ｈＥＧＦ基因克隆于测序正确的ｐＢＫ－信号肽双链中，挑选白色菌落进行ＰＣＲ及酶切鉴定。结果：（１）测序结果表明经ＰＣＲ扩增的特异性ＤＮＡ条带的基因序列与ｈＥＧＦ信号肽基因一致；（２）经ＰＣＲ及酶切鉴定获得了序列正确的ｐＢＫ－信号肽－ＥＧＦ真核表达质粒。结论：本法可构建ｈＥＧＦ基因真核表达质粒。

Objective:Systematic construction for eukaryotic expression of foreign genes. Methods:The human epidermal growth factor (hEGF) signal peptide gene was amplified from fetal kidney tissues using reversible transcription PCR technique,and then cloned into the PBK-CMV plasmid for sequencing. The amplified hEGF gene was cloned into the pBK-Signal peptide recombinant plasmid followed by PCR and enzymatic detection.Results : (1) DNA sequencing result showed that the amplified DNA sequence was identical to that of hEGF signal peptide gene. (2) The pBK-Signal peptide-EGF eukaryotic expression plasmid was obtained by PCR and enzymatic detection. Conclusion:The eukaryotic expression plasmid for human epidermal growth factor gene is constructed.