Construction and expression of eukaryotic expression vector harboring human neutrophil peptide 1 / 解放军医学杂志

ABSTRACT

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.