人中性粒细胞多肽1真核表达载体的构建与表达

目的 构建人中性粒细胞多肽1(HNPl)基因真核表达载体，为探讨HNP1基因工程生产的可能性奠定基础。方法 从健康人中性粒细胞中提取总RNA，用RT-PCR方法扩增出HNPl基因cDNA片段，将纯化PCR产物与pMD18-T载体连接，经酶切鉴定及测序确证，将其亚克隆入真核表达载体pcDNA3．1／V5-His-TOPO中．用脂质体转染CA3S-7细胞，用BA-ELISA法检测转染细胞上清中HNP1的表达。结果 从人中性粒细胞中克隆出人HNP1基因，经测序分析与GenBank公布的人HNP1核苷酸序列完全一致，并在COS-7细胞中获高效瞬时表达。结论 成功构建了真核表达载体pcDNA3．1／V5-His-TOPO／HNP1，为后续哺乳动物工程细胞株的建立奠定了实验基础。

Construction and expression of eukaryotic expression vector harboring human neutrophil peptide 1

Objective To explore the possibility of human neutrophil peptide 1 (HNP1) gene engineering, we construct the eukaryotic expression vector carrying HNP1 gene. Methods With RNA extracted from human neutrophil cell as template, cDNA encoding mature HNP1 was amplified by RT-PCR, and then it was inserted into vector pMD18-T. After restriction endonuclease digestion and DNA sequencing confirmation the gene was subcloned into eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO to construct a recombinant expression plasmid pcDNA3.1/V5-His-TOPO/HNP1, then the recombinant plasmid was transfected into COS-7 cells by lipofectamine, and the expressed product was identified by biotin-avidin enzyme-linked immunosorbent assay ( BA-ELISA). Results The sequence of HNP1 completely matched those published in GenBank, thus eukaryotic expression vector pcDNA3.1/V5-His-TOPO/HNP1 was constructed correctly. The ELISA results showed that the eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO/HNP1 could temporarily express HNP1 in COS-7 cells. Conclusion The successful construction and expression of pcDNA3.1/V5-His-TOPO/HNP1 pave the way for the stable expression HNP1 in mammalian engineering cells.